Cell culture, both primary and secondary, is central to studying the biological behavior of cells in the field of scientific research. Research in immune cells, such as naïve lymphocytes and stem cells, is vital for understanding the complex processes occurring in the body. In this context, the researchers in this article present an innovative cell culture system using a device known as the “Minimally Observable Bioreactor” (MOAB), which helps simulate the marrow-like environment for the cells’ natural states. Through advanced experiments, they were able to reveal rare tumor events in real time, paving the way for significant advances in cell culture science and gene therapy. This article discusses the details of the technology used, the main results, and the importance of these developments in cancer research and the treatment of immune diseases.
The Importance of Cultivating Primary Blood Cells and Stem Cells
The cultivation of primary blood cells and stem blood cells is fundamental in the fields of cellular biology and disease treatment, especially hematological disorders and tumors. The immune system is a vital part of the body and primarily consists of white blood cells produced in the bone marrow. The bone marrow directly contributes to the formation of immune cells, making it a significant focus of interest for scientists. The cultivation of primary blood cells opens new opportunities for research into gene therapies and tissue regeneration.
Current research tests the efficacy of blood cell cultivation for expansion and study purposes, such as a treatment for tumors and organ transplantation. The use of advanced technologies such as three-dimensional cell culture is essential for uniquely understanding cellular behavior, as these technologies provide more representative environments for the cells’ location in the body. For example, cultivating blood cells in complex systems helps identify how these cells respond to external influences, facilitating the discovery of diseases that can be analyzed under conditions similar to real biological phenomena.
Moreover, the challenges facing the long-term cultivation of these cells require new strategies, and one proposed solution is the use of advanced incubators such as genetically engineered bioreactors. This system allows for improving growth and research conditions, reducing the adaptation period required for the cells, and enhancing the process of monitoring developmental changes over time.
Challenges in Cell Cultivation and Tumor Cloning
Cultivating primary cells poses a significant challenge, especially when attempting to clone tumor cells. Cultivation processes for primary blood cells necessitate providing a suitable environment for adaptation and growth while efforts are made to detect changes that may lead to tumor formation. This environment requires not only the presence of nutritious components but also appropriate spatial organization, including fluid flow, which plays a critical role in ensuring sufficient nutrient supply and waste removal.
Researchers are developing small bioreactors, such as the MOAB Reactor, which mimic natural conditions to achieve extension and integrate advanced sensors to monitor cellular responses in multi-dimensional environments. Thanks to this, researchers can observe the behaviors of cancer cells, stem cells, and even embryonic stem cells. Research in this field is urgent to achieve breakthroughs in rehabilitation therapies and gene therapy.
Using small bioreactor designs makes it possible to reduce the adaptation time required for cells, which can take long periods in traditional systems. These systems enable scientists to monitor cell behaviors accurately, facilitating the discovery of abnormal growth conditions or transformations that may occur in the optimal growth environment and any changes in cell aggregates.
Cell Culture Techniques and Tumor Analysis
Advanced biological transformations in cells require a systematic approach based on the use of integrated cultivation systems. The techniques used include the integration of monitoring devices that continuously track cellular data, allowing researchers to detect changes in cellular physiology in real time. Through the use of multi-dimensional cell culture, including focal reactor systems, it becomes possible to evaluate the effects of unintentional genetic mutations on cell function.
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This system has the potential to provide fertilizers and nutrients in safe and reliable ways, facilitating better cell growth and gaining clearer insights into the effects of gene-editing methods like CRISPR. The ability of the system to encapsulate cells within an environment similar to that found in the body is vital for reducing the risks of undesirable events such as tumor formation. The system has ports for passing cultures and filters that improve the quality of the culture medium.
By relying on early analysis in such controlled cell culture conditions, researchers can distinguish abnormal events from normal cellular interactions. Using a reactor like MOAB, the behavior of stem cells can be studied with a focus on the early detection of cell growth abnormalities or stasis, contributing to identifying risks associated with gene therapies.
System Components and Dynamic Processes in Cell Culture
The processes related to cell culture involve using an advanced pump equipped with 20 cm long tubes of a specified inner diameter (ID = 1 mm) to ensure proper and monitored flow. These tubes are connected to Luer lock fittings and extend to 30 cm long oxygen tubes, forming a connection between the reservoir outlet and the MOAB (closed cell culture model) inlet. Another set of 30 cm long oxygen tubes, also fitted with Luer lock connectors, is used to connect the MOAB outlet to the gravity reservoir outlet. The pump operates at a rate of 0.3 revolutions per minute, equivalent to a flow rate of 18 microliters per minute, ensuring precise cell culture conditions.
When it comes to the cell culture environment, cells are maintained in dynamic conditions within a humid environment at 37 degrees Celsius with 5% carbon dioxide. A mixture of cells is prepared according to the required final ratios, and 100,000 cells are re-suspended in a final volume of 30 microliters of StemSpan™ SFEM media. These cells are directly seeded onto scaffolds that have been activated by fibronectin, using similar methods for manufacturing as mentioned in previous studies. After cell seeding, the scaffolds are incubated in a system called Petri-in-Petri, which contains three 60 mm Petri dishes placed inside a 150 mm dish filled with 1X PBS to maintain high humidity and reduce media evaporation. Through these meticulous processes, cell holders can achieve the best possible growth environment.
Circle Preparation Process and Media Change
The circle preparation process is the essential first step before sealing the covers with MOAB, maintaining the scaffolds’ humidity during extended airflow periods. This process includes refilling the reservoir with 7 ml of StemSpan™ SFEM media, activating the pump to increase its speed to 20 revolutions per minute until the media reaches the interface of the bioreactor chamber. After that, the pump is stopped, the speed is reduced to 0.3 revolutions per minute, and the covers are fitted to the main part of the bioreactor. At this stage, the system is placed next to the incubator, where the pump can be activated.
The media change process occurs every five days of continuous flow, where the pump is stopped, and the tubes are secured to prevent unwanted flow during the circuit handling. The system is opened under a sterile hood, and the consumed media is drawn from the reservoir outlet using a sterile syringe, followed by the addition of fresh media. After tightening all connections, the system is confirmed to have been returned to the incubator and secured with the pump. Once the fixation from the tubes is removed, the conditions are reinforced for cell care and to provide the best environment for development and replication.
Cell Viability and Growth Analysis
Cell viability tests are used to assess cell health through exclusion testing using the Trypan Blue dye, a common technique that shows the cells’ resistance ability. This method is used in numerous studies to measure cell regeneration. While cell growth is evaluated through analyzing cell amplification using flow cytometry, cells are fixed in 1 ml of PBS with the addition of 1 microliter of Cell Trace™ CFSE dye, facilitating the tracking of cell growth and division rates, enabling comparison of the performance of different systems.
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In addition, cell death is analyzed using flow cytometry with tools such as Annexin V staining for analysis, which helps understand the increase in death rates after exposure to harmful agents like staurosporine. Through these analyses, researchers can identify survival and growth mechanisms under complex and harsh conditions. All these tools help provide accurate data on cell health and efficiency throughout their cultivation period.
Isolation of Cells and Their Role in Experiments
Cell isolation is considered a critical step in cell research using effective methods. Immune granulocyte cells, such as CD4+ lymphocytes, are separated through a centrifugation process using the Ficoll-Paque® technique. CD4+ cells are enriched using magnetic cell separation techniques with a CD4+ cell isolation kit. Thus, pure cells are obtained and used in immunological experiments.
Isolation of CD34+ HSPC stem cells is carried out using similar methods, where they are extracted from umbilical cord blood for dual research efforts between cancer biology and regeneration. These cells play a crucial role in studies related to understanding cellular interactions and immune system responses. These steps are not only diverse but vital to providing diverse genetic patterns that can be used in upcoming experiments. The results of isolation bring rich data about cellular nutritional needs and interaction with new culture environments.
Gene Transformation and Use of Biotechnology
The techniques used in the transduction process, such as delivering genetic information to PLB-985 cells using viral vectors, are an essential part of modern research strategies. Using the spinfection method, important genes like GFP and mCherry are injected into cells. These processes enable researchers to track tumor cells and models derived from blood cells in a suitable experiment.
The process of selecting transformed cells involves using a FACS cell sorter to identify cells that contain the targeted genetic components. Research continues to improve production processes and genetic applications to achieve the desired benefits in modern gene therapies. These activities enhance the overall understanding necessary to assess the impact of genetic factors on cells for therapeutic purposes. These experiments will have a significant impact on developing effective treatments in various fields such as tumors, immune diseases, and tissue regeneration.
Modern Analytical Techniques and Cell Monitoring
The importance of analytical techniques in studying cell interactions under specific conditions is highlighted, where advanced microscopy technology is used to evaluate and discover cellular dynamics within tissues. Using the 3D Mikroskop TCS SP8, colored images can provide detailed data about cell states during different development periods, allowing researchers to understand how cells can interact with their environments.
This process implies great potential for real-time monitoring and diagnosing cells; cellular aggregates can be imaged, and contextual changes can be detected. These additional data help identify trends in growth and reproduction, providing information for informed decisions in developing therapies and research. These advanced techniques make it easy to test hypotheses and enjoy higher productivity in various cell cultivation and practical research.
Image Analysis Using Advanced Techniques
Image analysis is considered an essential part of biological research, where advanced imaging techniques are used to gather data and information about cells and tissues. In this context, a lens with a magnification rate of ×10 was used to capture detailed images of areas of interest. Subsequently, images at each time depth level were merged using the NIS-elements software, reflecting the importance of the interconnection between techniques to improve data quality. An important preliminary step in this process is taking care of preparatory aspects, such as enhancing contrast and removing backgrounds, to ensure improved image clarity before conducting the analysis. These preparatory processes lead to obtaining accurate data that provides deeper insights into cell behavior within specific environments, such as the silicon structures that were used in some experiments.
Systems
Stationary Culture and Its Influence on Cell Interactions
Studies indicate that stationary cultural systems play a vital role in understanding long-term cellular processes, especially in contexts such as tumor formation. Experiments have been set up to monitor the behavior of leukemic cells in environments containing normal cells. Observations derived from these experiments showed that the numbers of leukemic cells began to increase significantly while CD4+ lymphocytes were reducing their numbers over time. These results clearly indicate how weaker cells affect tumor spread, providing insights into the complex dynamics of cellular interactions. As experiments progressed, it became clear that stationary culture systems have particular limitations when considering long time periods, as the numbers of dead cells increase and cells begin to deteriorate, necessitating the search for more dynamic alternative systems.
Dynamic Models and Their Role in Understanding Tumor Growth
In light of the challenges posed by stationary culture, the dynamic culture model MOAB was adopted, which is an integrated system that allows for the maintenance of cell cultures over long periods. The system consists of key components including a peristaltic pump, a gravity reservoir, and a localized bioreactor. Such a system enables effective movement of the culture medium, increasing the efficiency of nutrient and substrate replacement for the cells. The reservoir was designed in a way that facilitates cell sedimentation at the bottom, thereby enhancing the effectiveness of the pump’s suction. Cultural models were aligned with a three-dimensional tissue model representing the bone marrow environment, reflecting how dynamic models are used to explore complex cellular interactions. The three-dimensional model was introduced to showcase the varying effects on cell growth and the ability to simulate natural conditions far from traditional models that were difficult to utilize.
Establishing Criteria for Detecting Cancer Cells
To seek models and detect cancer cells, it was essential to prepare a suitable culture environment that aligns with the requirements of lymphocytes and blood-forming cells. An appropriate culture medium was provided using a mixture of two media, and the leukemic cell medium was gradually adjusted until a uniform state was reached. This process indicates the importance of removing discrepancies between types of culture media to enhance biological experiments. Leukemic cells were used as a benchmark for detecting growth experiments, allowing for the assessment of their behavior in the new medium. These cells underwent multiple tests to determine their adaptability and growth, revealing that their performance remained consistent in both environments, enabling their use as an indicator of cancer cell growth.
Conclusions Drawn from Exciting Experiments
All these articles and experiments contribute to understanding how cells and environmental factors influence tumor development and growth patterns. Researchers continuously strive to improve cultural models to produce results that reflect the accurate realities of natural life. Despite the challenges of using traditional systems, techniques such as dynamic culture provide notable advancements in the research world, facilitating the discovery process of tumors and cell interactions. These modern systems could play a pivotal role in designing new methods for disease treatment and prevention, allowing us to explore new horizons in biological sciences.
Manipulating Cancer Cells and Utilizing Bioreactor Systems
Cancer cells present a significant challenge in the fields of medical research and treatment, and many systems have been developed to study these cells and understand their behavior. Among these systems is the MOAB (Micro-Organ Culture System), which has been used to test PLB-985 cancer cells. In the context of the experiments, cancer cells were modified to introduce stable expression of green fluorescent protein (GFP), leading to the production of new strains that facilitate the study of cell behavior under laboratory conditions. This system aims to achieve precise analyses of the effects of environmental factors on cancer cell behavior, which may contribute to improving treatment strategies.
The system
The user allows for the imaging and identification of a minimum number of cancer cells, which has been defined in this study as 100 cells. Flow cytometry results showed that the studied cancer cells were able to survive and grow under these conditions, reflecting the importance of focusing on their protection in a dynamic environment such as that provided by the MOAB system.
Assessment of the Detection Capacity of Cancer Cells Under Cooperative Conditions
Experiments were conducted to evaluate the MOAB system’s ability to define and detect cancer cells in the presence of CD4+ lymphocytes. 100 cancer cells were mixed with 100,000 lymphocytes and seeded in a scaffold made of silk fibers supported by fibronectin. After a period of time, it was observed that the cancer cells were able to grow significantly, while the lymphocytes remained clear in the environment. Fluorescent imaging techniques were used to monitor the development of these cells over time.
Moreover, the cloning process among the lymphocytes was measured, showing that a portion of the CD4+ cells were active and accumulated while another portion reflected lower levels of activity. This data provides accurate insight into how cancer cells interact with the immune system, which can be of great importance for improving immunotherapies and cancer drugs.
Sustainability of Immune Cell Life in Dynamic Environments
One of the main goals in cancer research is to enhance the availability of immune cells to protect the body from tumors. The MOAB system provided exciting data illustrating the capacity of CD4+ lymphocytes to survive and maintain activity over a period of 3 months of dynamic culture. The analyses revealed interesting results, observing that tumor cells exhibited a significantly higher growth rate compared to immune cells, indicating that the system provides a favorable environment for other cell types, including tumor cells.
This data is vital for understanding the biological processes that influence the efficacy of immunotherapies, and the MOAB system reflects how rates of proliferation and changes in cells can be tracked in dynamic environments. This is also an important starting point for further research on how to develop more effective therapies that take into consideration the interaction of cancer cells with their surrounding cellular environment.
The Potential of the MOAB System in Cell Development and Genetic Research
The exploitation of the system in stem cell research and the importance of locating tumor cells in the internal environment of the body represents a promising area. By creating an environment similar to that of bone marrow, the system’s performance was evaluated with three categories of cells including cancer cells, resulting in exciting outcomes regarding the proliferation of cancer cells and the onset of stem cell deterioration. This shows how similar ecological systems can be useful in understanding the mechanisms of cell interactions.
The results suggest the potential to improve therapeutic approaches by targeting models that reduce the phenomenon of healthy cell deterioration in the presence of infiltrating cancer cells. Utilizing this system is an opportunity to enhance research directions and explore new techniques in gene therapy.
Cell Culture Experience: Methods and Challenges
Cell culture is a fundamental technique in the fields of medical biology and clinical research. The importance of these systems lies in their ability to simulate the natural environmental conditions for cells, leading to a deeper understanding of their behavior and functions. Among the significant challenges facing scientists when culturing certain cells, especially T cells and hematopoietic stem and progenitor cells (HSPCs), is maintaining their viability and preventing them from differentiating in unsuitable culture environments. In this context, quality culture protocol systems have been developed to sustain the cells and facilitate their study. These systems offer the opportunity to study the complex interactions between different cell types, especially in the presence of cancer cells alongside healthy cells.
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Our cell culture systems emphasize the importance of various environmental factors, including growth and nutrition, as well as the continuous flow of stimulating factors. By applying these methods, it was observed that stem cells suffer a continuous decline in their numbers over time, which is consistent with differentiation and exhaustion. In contrast, cancer cells showed significant proliferation, indicating that the life continuity of stem cells can be maintained to some extent while supporting the ongoing growth of tumor cells. This type of understanding provides valuable insights into the dynamics of cancer evolution.
Modern Technology: The Milli System and Its Benefits
In recent years, new systems such as the MOAB system, which relies on Milli-Filtration setups, have been tested. This technology provides a unique environment that enhances the interaction between multiple cell types, facilitating the expansion of tumor cells. Studies have shown that these systems support the retention of normal cells while simultaneously allowing tumor cells to proliferate. This indicates the potential to identify rare tumor cells in critical cases in line with therapeutic applications.
One of the main features offered by the Milli system is its ability to provide a continuous flow of stimulating factors, enhancing growth and proliferation. The system’s design allows for the cultivation of a large number of cells in each chamber and also enables monitoring of tumor cell variability in a controlled environment. This represents a significant achievement in cancer disease research, as the data derived from such experiments can directly contribute to understanding how tumor cells can evolve and proliferate under certain conditions. Additionally, improving the ability to recognize rare tumor cells enhances the effectiveness of therapeutic interventions.
Research and Development Applications: The Importance of Genetic Profiling
Genetic analysis is a crucial step in understanding how cells evolve and distinguishing between healthy and cancerous cells. Modern techniques such as single-cell RNA sequencing (sc-RNA seq) provide unprecedented opportunities to study gene expression at an individual level. The ability to analyze gene expression patterns accurately sheds light on the mechanisms that cells use to interact with their surrounding environment and enhances our understanding of vital biological processes such as differentiation and growth.
In current research, the MOAB system has been used to evaluate gene expression data from different cells, aiding in the identification of the timing and intensity of genetic changes occurring in tumor cells. This information can serve as a starting point for identifying new targets for early treatment or preventive interventions. It becomes clear that an increased understanding of these processes requires advanced technology and interdisciplinary collaboration that combines molecular biology, genomics, and cancer science.
Future Challenges and Tests
Although the MOAB system presents new possibilities, significant challenges remain. Among these challenges is the difficulty of conducting comprehensive tests regarding the availability of live cells and their response to external factors. Due to the three-dimensional complexity of the system, conducting accurate analyses becomes challenging, necessitating the development of new analytical methods and innovations to enhance the efficiency of this process. These issues are of paramount importance concerning the applicability of future research and therapeutic developments.
Furthermore, it remains essential to enhance our understanding of the dynamics of stem cells and tumor cells, including the influence of external factors on their behavior. These aspects will remain fundamental to cancer research and help science move toward more effective therapeutic forms. Future experiments based on adaptable systems may serve as a platform for analyzing complex issues and genetic diversity in cells under conditions close to natural growth.
Challenges
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In Developing Positive Control Based on CD34+ Cells
In the field of stem cell research and gene modification, developing a reliable positive control system is crucial to ensure the safety of gene-engineered therapies. However, our attempts to develop this system have faced numerous challenges. One of the main factors is the phenomenon of aging caused by oncogenes, where these genes promote aging processes in the liberated cells. Essentially, this leads to significant difficulty in producing cancerous cells from primary human cells, making the process exceedingly complex and necessitating millions of cells for replication. These limitations highlight the urgent need to develop non-invasive tests to measure cellular efficacy that correspond to three-dimensional structures.
Unfortunately, those attempts to achieve the development of a successful positive control system have not succeeded, highlighting the challenges associated with leveraging genetic engineering in clinical applications. Therefore, we must open new research areas aimed at improving this system, which requires exploring methods that could contribute to developing more accurate and cost-effective tests for detecting oncogenic transformations in genetically modified stem cells.
MOAB Model as a Means to Detect Tumor Transformations
Based on the challenges facing traditional control systems, the MOAB model has been introduced as a new approach enabling researchers to facilitate the culture of primary human cells, such as CD4+ lymphocytes and red blood cells. This model is a significant step forward, as it allows for the detection of rare tumor cells present in the bloodstream, opening the doors for a better understanding of tumor development and treatment response. In this context, at least 100 cultivated cells can be detected, which is a much smaller number compared to traditional systems such as the soft agar system.
This system could have a significant impact on how the safety of gene therapy techniques is evaluated before they transition to clinical trials. Given the paramount importance of patient safety, the early detection of any undesirable tumor transformations could help guide future research and improve safety in performing transplants of modified stem cells.
Future Prospects for the MOAB Model and Clinical Assessments
In light of the current model’s success and potential effectiveness, there are exciting prospects for adding additional models in other chambers of the bioreactor, where a related environment to the modified cells could be recreated. This may aid in studying the interactions of these cells with their spatial environment, encouraging better understanding of how cancer develops and how various treatments affect it.
This represents a new pattern in research methods, moving away from traditional models that are often unsafe or unreliable. As development of these research tools continues, the opportunities to expand this system become clearer. Continuous efforts are required to enhance research and development to achieve the scientific standards necessary to translate results from laboratories to clinical applications. In the future, this system may also lead to the development of new metrics to measure the effectiveness of therapies and clinical applications, benefiting a wide range of patients.
The Importance of Ethical Steps and Funding in Scientific Research
Ethical steps and funding play a crucial role in the sustainability of scientific research, especially in sensitive areas like gene modification and cell-based therapies. Working with human cells requires precise ethical approval, which has been confirmed in this research according to local regulations and institutional requirements. Synchronizing research with ethical considerations ensures that the fundamental goal of scientific research is not lost: to serve humanity and improve quality of life.
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Funding has played a key role in the success of this research. Grants funded by entities such as the Replacement, Reduction, and Refinement (3Rs) Center, and other governmental bodies, enable researchers to advance ambitiously in their studies. The presence of sufficient financial support contributes to opening new horizons for innovative ideas and enhancing future therapeutic applications.
Collaboration and Communication in Genetic Engineering Studies
Collaboration between researchers and practitioners in scientific communities is a fundamental pillar for achieving progress in genetic engineering research. The exchange of knowledge and experiences enhances the effectiveness of proposed solutions and addresses current challenges more swiftly. In this regard, building networks and partnerships between academic and industrial institutions is particularly important, especially when focusing on clinical applications. By working together, researchers can develop new strategies and explore unexplored areas to enhance safety and effectiveness in gene-modification-based therapies.
Gene Therapy and Its Effects
Gene therapy represents one of the modern medical initiatives aimed at treating hereditary diseases by altering the genetic material of patients. Despite its tremendous potential, this approach is a controversial topic due to potential risks. Several studies have shown that gene therapy may lead to serious side effects, as occurred in the case of X-linked severe combined immunodeficiency, where the introduction of genes resulted in unwanted conditions that led to tragic side effects.
Discussions around this issue revolve around the balance between the potential benefits of correcting genetic defects and the side effects that may include negative impacts on the immune system or the onset of diseases such as cancer. Therefore, it is essential to instill awareness and conduct a thorough evaluation of gene therapy pathways, while monitoring potential side effects at the cellular and genetic levels.
As part of these studies, the role of stem cells has been analyzed, where recent research has indicated that using stem cells in rehabilitation can represent a significant step toward achieving effective therapy. However, caution must be exercised in handling these cells, as the function of genes controlling their growth and differentiation plays a vital role in the success of medical applications.
According to certain studies, inaccurate manipulation of genes within stem cells can increase the risk of tumor growth. Thus, continuous monitoring of stem cell interactions and understanding genetic links are crucial to avoid those risks.
Cell Cultures and Their Role in Medical Research
Cell culture fields, whether two-dimensional or three-dimensional, are vital tools in medical research. These cultures represent vital models for many clinical studies, allowing researchers to understand how cells interact under various conditions and to address the complex properties of living tissues.
Studies indicate that three-dimensional cultures provide an environment that is closer to actual conditions within the body, facilitating the analysis of cell behavior. This approach can be very beneficial in studying phenomena such as cell migration and immunity, and allows for the study of mechanisms by which cells respond to treatment.
When comparing two-dimensional cultures to three-dimensional ones, the clear benefits of the more complex models emerge. Three-dimensional cultures can mimic the way natural tissues are organized, which is advantageous in studying cancers and their response to treatment. They can also shed light on how environmental factors like matrix stiffness affect cell behavior.
Modern cell culture techniques involve using biological materials and genetic techniques to make the models more accurate, revealing intricate details about cellular interactions. When combined with advanced imaging techniques, it is possible to gain invaluable insights into disease progression.
Challenges
The Challenges Facing Biotechnology and Rehabilitation
While it appears that fields of biotechnology play a key role in developing new therapies, there are challenges that hinder the progress of these fields. One of the most prominent challenges is the difficulty of simulating the precise conditions found in living tissues when developing tissues or organs. The materials used in their fabrication may face issues of biocompatibility or an unforeseen appearance that renders them ineffective.
One study suggests using 3D models to mitigate these risks, as these models allow for a better understanding of cell interactions with various environments. However, producing and designing these models requires resources that are not always available, complicating research and development processes.
Laboratory experiments show that maintaining the stability of cells and tissues in controlled environments is difficult to achieve and often requires the use of highly advanced techniques. Additionally, there are complex requirements for closely regulated clinical trials to ensure successful experiments, reflecting the need for improved research quality and healthcare services.
Furthermore, advancements in technology must be balanced with ethical considerations related to new treatments. Research must be transparent to the public and adhere to ethical standards that reflect societal values. Continuous interaction between scientists, improved educational platforms, and community engagement can contribute to achieving this.
Development of 3D Systems in Cell Cultivation
Cell cultivation in two-dimensional environments is essential for understanding cellular biology; however, it lacks the ability to model the complex environmental conditions associated with living organisms. 3D systems provide an alternative solution by allowing the creation of a more representative environment of physiological conditions. These systems enhance cell interaction and increase the potential for cellular scalability. In 2D environments, cells face growth limitations due to the lack of continuous flow of the culture medium, posing a challenge in supplying nutrients and removing waste. Thus, 3D systems are more beneficial in fields such as tissue engineering, drug discovery, and cancer research.
One advanced model in these systems is the “Micro-Optically Accessible Bioreactor” (MOAB), which allows for the cultivation of three-dimensional cellular structures under a continuous flow of culture medium. By providing an environment similar to that found in living tissues, research in this type of reactor can achieve significant steps toward understanding cell behavior in a natural environment. For example, these environments may allow for the processing of certain types of cells, such as granulocytes or lymphocytes, and exploring how these cells respond to treatment or radiation.
Challenges in Cultivating Blood Tissue Cells and Solutions Strategies
Live tissue engineering, especially in cultivating blood tissue cells, faces several challenges related to cell growth and their surrounding environment. Blood tissues, such as bone marrow, are highly complex and rely on supportive structures and their surrounding medium. Their functions are controlled by hematopoietic stem cells (HSCs) which represent the fundamental seeds for the development of all types of blood cells.
The sustainable process of cell cultivation requires precise models that mimic the environmental conditions of the bone marrow. For example, certain systems such as silk-based bioreactors that can simulate the natural physiology of bone marrow have been introduced. These systems promote the stimulation of platelet and stem cell production, enabling medicine to achieve tangible progress in the transfer of stem cells used for treatment.
Moreover, the integration between stem cell transfer and allowing for genetic infiltration or gene therapies requires the development of systems to identify potential adverse events such as tumors. Traditional methods of tumor detection rely on animal models and often prove ineffective in recognizing pre-tumor events. Customizable systems provide a higher degree of precision that helps reduce the risks associated with genetic modifications.
Impact
CRISPR Technology in Blood Tissue Cell Cultivation
The control of gene editing techniques, such as CRISPR, lies in their exciting potential to correct genetic mutations. Despite their benefits, there are specific risks associated with these modifications, including the dormancy of malignant mutations that may develop post-procedure. Laboratory cultivation systems – like the mini bioreactor – provide a valuable model for monitoring the development of genetically corrected cells and the potential for cancer transformation, offering a suitable platform for such studies.
By integrating cell culture with precise monitoring of biomarkers, unstable biological entities can be challenged, enhancing the quality of gene therapies, and ensuring patient safety. These effective systems offer a new approach to designing tissues and genetic modifications with greater precision. As research advances, new means will attempt to address the issues related to the safety and general efficacy of cell transfer.
The model developed using MOAB may secure an environment that scientists have long sought to understand primitive cellular mechanisms and their relation to diseases and the mysteries of smart tissues. This model presents a new horizon for future research to study tumor-specific factors and the safety of innovative therapeutic techniques.
Experimental Results on Cell Cultivation and Cancer Monitoring
In experiments conducted on MOAB cultivation models, the validity of this system in effectively growing real blood cells was verified, allowing for monitoring their growth and demonstrating their reproductive abilities. The results obtained show that the system supports an ideal environment for cells such as CD4+ lymphocytes and CD34+ hematopoietic stem cells, with the potential to attract cancer cells like those derived from leukemia cell lines.
Through the experimental study, researchers succeeded in observing how tumor cells grow and extend in specific environments, contributing to the classification of evidence on the evolution of malignant tumors. These results are positive and pave the way for further investigations on how genetic modifications and environmental models affect the safety of cellular transformation.
Scientists assert that this dynamic unit is not only an effective means for cultivation but should play a vital role in modern healthcare, as it can be used to design and test gene therapies tailored to the needs of individual patients, enhancing the delivery of more precise and effective treatments.
Techniques Used in Cell Cultivation
Cell cultivation techniques involve several complex steps aimed at improving and enhancing the efficiency of cell growth and testing effectiveness. The program begins by suspending the cells in an effective cultivation medium, where mixed cells are prepared at certain concentrations and then frozen in a new medium, ensuring an optimal environment for their growth. The cell cultivation technique uses carriers treated with Fibronectin, facilitating cell adhesion to them and ensuring their continued growth within a trained system to provide optimal growth conditions. This type of carrier is particularly useful in stem cell research.
After the planting process, a high level of humidity is maintained to avoid drying out the cells. Therefore, the Petri-in-Petri technique is used, where dishes of different sizes are utilized to maintain a moist environment. The dishes are placed under water pressure to facilitate field tests in the laboratory.
At the point where cells are seeded, they are allowed to grow for 72 hours before applying stresses or new media, allowing sufficient time for nutrient exchange.
Cell and Vitality Analyses
It is essential to conduct tests to evaluate cell vitality and growth. The Trypan Blue dye test is used as an indicator of cell vitality, as the dye penetrates dead cells without affecting live ones, thus facilitating immediate assessment of the ratio of live to dead cells. This data is crucial in testing the effectiveness of various treatments.
The technique for measuring cell proliferation using flow cytometry is also employed, where a specific dye is added to distinguish live cells from dead ones. Samples of cells are taken after treatment, and their distribution and concentration are measured, which helps determine any changes in cell numbers over time. These examinations are considered necessary to determine the effectiveness of growth and cellular differentiation.
Planning
Preparation of Systems for Cell Culture
Planning complex biological systems requires precision in preparing and equipping the system components. The technology of “Circuit Priming” is used to prepare the system before connecting it to the carriers, preventing drying and allowing liquid flow evenly. The pump speed is controlled to ensure that the medium reaches the required extremities within the culture system.
After the preparation process, a medium change period is set every five days, where the remaining medium is replaced with new medium after ensuring that all connections are secured. This change is a fundamental part of maintaining an ideal cellular ecosystem and enhancing growth efficiency.
Evaluation of Apoptosis and Cell Isolation
Measuring natural cell death (apoptosis) is essential for understanding how cells respond to different treatments. These measurements are conducted using flow cytometry strategies, such as the use of Annexin V stain and examining cells using cytometric flow techniques. Excellent tools are used to measure the rate of cell death and differentiate between live and dead cells.
On the other hand, cell isolation, such as CD4+ T lymphocytes, plays an important role in immunological studies. Magnetic enrichment techniques and cell sorting devices are utilized for this purpose. This isolation is very effective for use in clinical and therapeutic research to develop targeted treatments.
Testing and Assessment Using Advanced Imaging Techniques
The leading research steps involve using advanced imaging techniques to analyze cells in different environments. A fluorescence microscope and high-resolution light examination are used to analyze samples taken from the culture medium. This imaging allows for obtaining enlarged and colored views, aiding in studying cell behavior and formation over time.
Moreover, imaging techniques enable researchers to analyze cell distribution and determine the presence or absence of specific markers within cultures, contributing to accurate results that reflect cell behavior under certain conditions. This type of analysis can have profound implications for understanding complex cellular processes and biological systems in general.
Dynamic Cell Culture Systems and Tumor Event Estimation
Dynamic systems are essential for understanding cellular processes in the long term, especially those related to tumors. Current studies represent significant progress in assessing tumor activity through cell culture in dynamic systems, allowing for accurate information about cellular behavior and interaction among different cell types. Traditional tumor tests such as soft agar colony formation assays and three-dimensional cell cultures have been relied on; however, these methods do not suit blood and lymphatic cellular tissues well. Therefore, a new method based on cultivating a complex mixture of primary cells was developed, allowing highlighting tumor events accurately. In the research, leukemia cells of type PLB-985 were used with CD4+ T cells as a social sample.
During the research, it was observed that the number of CD4+ T cells decreased over time, while the number of leukemia cells increased, reflecting the gradual expansion of the tumor population. However, static systems appear to have limitations in their ability to assess the tumor explosion over the long term, as an increase in the number of dead cells and a significant decrease in cell motility were recorded. Thus, these systems revealed their inability to contain live cells for long periods, necessitating consideration of adopting more effective dynamic culture systems.
Designing and Manufacturing Dynamic Systems
In an effort to improve cell culture systems, a MOAB (Miniature Open Bioreactor) device was used to facilitate long-term cell cultivation. This system includes key components such as a axial pump and gravity actuator, where the culture medium is effectively drawn and pumped through the MOAB. The design of this device ensures the maintenance of oxygen concentration in the medium, which is a critical factor for sustaining cell life during cultivation. This system operates dynamically, allowing control of medium flow to ensure optimal growth conditions.
All
The MOAB reactor contains three chambers, which can be connected in series or parallel. These chambers are designated to contain a functional substrate of fibronectin, reflecting a bone marrow-like environment. Experimental results have shown that this dynamic system improves cell survival and growth rates compared to static systems. Despite harsh culture conditions, this innovation ensures accurate and useful results in cancer-related research.
Establishing Tumor Cell Detection Index
After constructing the dynamic culture system, it was essential to create new protocols that define tumor cell growth. The first steps involved using the leukemia cell line PLB-985 and preparing it to grow in the common medium, Stem Span™ SFEM. Over several weeks, the medium concentrations were adjusted to achieve complete acclimatization of the cells. After this process, the cells showed significant improvements in growth and tolerance. Analyses were then conducted in different culture settings to confirm that the new conditions did not adversely affect cell characteristics, such as growth and death rates.
Cellular response testing was conducted using flow cytometry techniques, which showed no significant difference in the proliferation rate between cells cultured in RPMI and those in Stem Span™ SFEM. These results provide a standardized benchmark for estimating tumor cell growth, facilitating the evaluation of treatment efficacy and the search for new therapeutic targets. While numerous experimental results indicate a promising trend for future utility in medical research and cancer treatment.
Potential Implications of Current Discoveries
The results derived from the dynamic systems and tumor cell evaluation mechanisms hold immense potential for applications in clinical research and targeted tumor therapies. The advanced capabilities of these systems are valuable for assessing tumor cell response to various treatments and mapping the interaction between immune and cancer cells, which could support the development of new therapeutic strategies. Furthermore, utilizing small bioreactor technology may contribute to understanding the mechanisms of tumor spread and resistance to treatments, which is a crucial step towards improving clinical outcomes for patients.
It is also important to note the necessity of continuing to develop these systems and increasing their adaptability to different conditions, such as various types of tumors or any changes induced by treatments. This type of research enhances the scientific community’s ability to arrive at innovative solutions to complex medical problems, ultimately leading to an improved quality of life for patients suffering from tumors. In the coming years, it will be essential to continue research in this area, reflecting the importance of dynamic systems in biological and cancer research.
Characterizing Cellular Patterns in Complex Tissue Environment
When studying cellular phenomena associated with cancer, it has become crucial to use advanced techniques to detect cancer cells in multicellular environments. In this context, a system has been introduced that is effective in detecting cancer cells in a precisely prepared tissue environment. The system employs dynamic culture characteristics, determining the minimum number of cancer cells that can be cultured while maintaining a ratio of viable cells, facilitating the study of interactions between cancer and immune cells. Initial results showed that only 100 cancer cells could exist for an extended period, which is an achievement compared to traditional requirements that often demand around 5000 cells.
Identifying cancer markers in a complex biological milieu such as the lymph node environment is vital for understanding how cancers develop. By conducting a 15-day culture, cellular patterns were effectively identified. Various sensitive techniques were utilized to monitor the results of this cellular interaction, aiding scientists in understanding cancer behavior under conditions akin to those in the body. This allows researchers to study tumor growth dynamics and interactions with immune cells more accurately, which is a crucial aspect often lacking in many other study models.
Interaction
The Interaction Between Cancer Cells and Immune Cells
The interactions between cancer cells and immune cells are topics of significant interest in cancer research. Various areas related to the relationship between cancer cells, such as PLB-985, and immune cells, such as CD4+ T lymphocytes, have been explored. Experiments have been designed to allow researchers to observe the dynamics of this interaction. Studies have shown that cancer cells can grow substantially while the number of immune cells remains constant, indicating that cancer cells exploit certain mechanisms to survive and achieve growth.
The experiments involved mixing 100 cancer cells with 100,000 immune cells, observing how each type evolved over a period of up to 3 months. This represents a suitable in vitro model for studying how the nature of immune interaction changes as tumor cells continue to expand. Through visual monitoring and advanced techniques, the differentiation of immune cells and their density changes with the growth of cancer cells could be observed, reflecting the challenges the immune response faces in combating cancer.
Identifying Rare Tumor Events in the Bone Marrow-like Environment
Building a tissue-like environment similar to bone marrow is an important goal in stem cell studies and gene therapy. The MOAB system was included in the experimental procedures to evaluate its performance in detecting cancer cells alongside stem cells. In this experiment, cancer cells were incorporated with human stem cells to create an environment that mimics bone marrow characteristics. The results were striking, showing a clear increase in the number of cancer cells against a decrease in the number of stem cells. This reflects how cancer cells influence the surrounding tissue context and the changes they induce in stem cell composition.
These findings raised questions about how to maintain the effectiveness of stem cells within a laboratory setting, paving the way for further explorations. Over a continuous cultivation period of 3 months, it was determined that cancer cells continued to divide rapidly, while stem cells were adversely affected and exhibited a decline in their numbers. These unbalanced dynamics are part of the discussion on how immune therapies can address these types of tumors and how the effectiveness of stem cells can be enhanced as a treatment option.
The Importance of Stem Cell Cultivation and Its Relation to Cancer Diseases
Stem cell cultivation is one of the vital areas highly regarded in modern medical research, especially concerning cancer treatment and the increasing developments in gene therapy approaches. Stem cells, particularly CD34+ hematopoietic stem cells, are a central focus in this field, as these cells retain their ability to renew and differentiate into various blood cell types. However, cultivating them in laboratory conditions remains a significant challenge that requires advanced techniques to ensure protection against the loss of stem cell properties, which directly impacts the efficacy of potential therapies.
Stem cell cultivation requires a specific environment and precise stimuli to maintain their continuity, including growth factors and cytokines. Especially when dealing with CD34+ stem cells, it is crucial to preserve their undifferentiated state to facilitate their use in therapeutic applications. Thus, the MOAB cultivation system presented in the research plays a crucial role in enhancing the sustainability of hematopoietic stem cells and their ability to expand. This aids in studying microscopic cellular behaviors and monitoring the interplay between stem cells and cancer cells, highlighting the importance of this system in early detection experiments for cancerous transformations.
The MOAB System and Its Capabilities in Detecting Cancer Cells
3D microscopy systems like the one listed here with MOAB represent a significant advancement in cancer research. This system allows for the cultivation of hematopoietic stem cells and CD4+ lymphocytes for extended periods without activation, making it an effective tool for investigating cellular effects without the numerous external interferences that may arise when using traditional cultivation methods. Consequently, this system allows for accurate assessment of the effects of genetic manipulation on cells, contributing to the identification of cancer-prone changes.
It provides
The system also has a positive feature, as it enhances the natural selection of cancer cells among normal blood cells, allowing for their unselective expansion, which may help in the detection of rare cancer cells that would have gone unnoticed under traditional cultivation methods. These new capabilities provide greater opportunities to study the genetic sequencing and cellular evolution of cancer cells, representing a significant leap forward in early cancer detection and identifying areas that may cause cancerous transformations.
Challenges and Limitations Associated with Stem Cell Cultivation in 3D Environments
Despite the promising potentials of the MOAB system, there are many challenges facing the cultivation of stem cells in three-dimensional environments. Among these challenges is the difficulty of conducting comprehensive analyses of cell safety and cell flow analysis. Retrieving live cells from the supporting matrix for molecular analyses can be complex, leading to difficulties in monitoring cellular behavior and analyzing various characteristics.
Recovering cells trapped in the supporting matrix requires advanced techniques to overcome these obstacles. The recovery of embedded cells in 3D implants requires highly sensitive mechanisms to retrieve those live cells and to monitor them optimally. However, the system offers the possibility of recovering retrieved cells by changing the medium, allowing for a significant percentage of recovered cells, but retrieving cells that have been retained for long periods remains a barrier hindering the analysis process.
Additionally, critical imaging techniques face challenges due to light interference and interference caused by biological materials in the overall structure, making it difficult to detect important biological indicators. These elements strongly indicate the need to improve the methods used to study cancers comprehensively within three-dimensional structured materials without affecting the quality of the results that research aims to achieve.
Future Trends and Innovations in Stem Cell Cultivation
The current research findings reflect the need for continuous innovation in cell culture techniques. There is an urgent need to expand the understanding related to improving stem cell culture conditions, and developing non-invasive tools and sensors compatible with 3D. Genetic codes and other factors affecting genetic transformation are central to understanding potential carcinogenic processes, in addition to providing opportunities to study and analyze genetic effects in greater detail.
Developing systems focusing on non-destructive testing procedures is among the biggest future trends. Such systems could contribute to the immediate and accurate verification of cell responses, facilitating the assessment of gene therapy efficacy and significantly shortening cancer transformations. Through advanced techniques, it is possible to maximize area and therapeutic desires, allowing for more successful and accurate treatment outcomes.
Continuous innovation in how stem cells are handled enables access to new and effective ways to treat cancer diseases and improve research related to the immune system. Research is moving towards the use of small-sized boxes equipped with advanced technologies to detect genetic patterns and detailed information about cultivated cells, representing a key step in developing new and effective treatment methods to combat cancer.
Model for Evaluating the Safety of Genetically Modified Human Stem Cells
The model is based on a system called MOAB, which aims to study the effects of genetic modification on human stem cells. In this context, blood stem cells are obtained from patients suffering from monogenic diseases such as sickle cell disease or thalassemia, or severe combined immunodeficiency. The genetic modification process is carried out to correct the gene responsible for the disease. After that, these cells are tested in the new system to detect any undesirable spread of cancer cells. These steps are crucial to confirm the safety of the techniques used in genetic modification, as any discovery of spread indicates that the technology is not safe for clinical use.
The model
The proposal includes multiple environments within the bioreactor, allowing for testing how modified cells interact with different simulated environments. The model recreates the natural environment where the altered cells exist, helping to provide deeper insights into how these cells transform into cancerous states.
If the test shows no significant increase in the number of cancerous cells, autologous cell transplantation (cells taken from the same patient) becomes possible. However, in the case of discovering malignant behavior, the use of these cells for transplantation is prohibited, reflecting the importance of safety in clinical trials.
Expansion of the System and Future Applications
In the future, it may be possible to expand the safety assessment system to include more models or methods that enhance understanding of the effects of genetic modification. Including additional models within the bioreactor is one of these promising options. This can provide a more complex analysis of environmental impact factors on modified cells. For example, models simulating different biological environments could be used to model the effects of attraction factors on the cells. This may help improve cell transplantation outcomes and enhance the body’s response to treatment.
It is noteworthy that ongoing research is essential to develop new techniques that can complement or improve the current model. Through advanced genetic modification processes, new solutions can be adopted for more effective treatment of intractable diseases. Refining bioreactor models may lead to more accurate results in detecting cancerous cells, contributing to the development of effective treatments based on genetic modification.
System Efficiency in Detecting Tumor Cells
The new system demonstrates a remarkable efficiency in detecting rare cancerous cells, as it can detect small amounts down to 100 implanted cells, significantly reducing the quantities required compared to traditional methods like soft agar systems. This means that the system has the potential to enhance cancer research, especially in the early stages of the disease where cells are less pronounced.
The broadening of this model to include two-dimensional and three-dimensional systems enables it to better simulate the biological environment, allowing for more precise analysis of cell interactions. The accurate model can provide insights into how cancerous cells grow and how they interact with potential treatments. With continued development, the system is expected to become a cost-effective and sensitive tool for tumor monitoring, providing an accurate assessment of outcomes after genetic treatment.
Ethical Data and Research Support
Ethical data is an integral part of any medical study or research. In this case, ethical approval for the study on humans was not required. This study came in response to local legal requirements and relevant research institutions. Obtaining data directly from patients or volunteers is part of the healthcare and intensive research process.
Additionally, research teams provide additional resources and supporting data for the results achieved, as collaboration with specialized centers and doctors enhances the quality of this research. Transparency in data handling and the presence of multiple funding sources help effectively support the progress of the research, facilitating the application of further clinical trials as a step towards broader implementation of genetic therapies.
Three-Dimensional Cell Technology
Three-dimensional cell technology is one of the modern innovations in cell biology, providing a more accurate model for simulating the physiological conditions of the body. This technology relies on creating three-dimensional structures that represent tissues and organs better than traditional two-dimensional models. Through this methodology, studying cell interactions and communications can be approached more objectively, enhancing the understanding of phenomena such as cellular proliferation, migration, and other vital activities.
Contributes to
3D cell models in the development of new drug tests enable scientists to assess the effects of drugs on tissue cells more accurately. For example, 3D cell systems have been used to study tumor responses to drugs, contributing to improved therapeutic outcomes for cancer patients. These models have also been utilized in research for cell-based therapies, necessitating the development of new culturing techniques that support these complex tissues.
One of the challenges facing this technology is the limitations of oxygen delivery to cells in the tissues, as oxygen plays a crucial role in cellular metabolism and growth. Developing better systems for oxygen delivery is an important step to avoid problems associated with advances in cultured tissues. These developments will enhance the 3D cell systems and allow for more complex and significant research.
Challenges Associated with Cancer Treatment
Cancer treatments have seen significant advancements over the past two decades; however, challenges remain. One of the biggest challenges is treatment resistance, as cancer cells can adapt and evolve to be resistant to the drugs used in therapy. Many mechanisms contributing to this resistance have been identified, including modifications in gene expression and intracellular signaling pathways.
Addressing this challenge requires a deep understanding of cancer cell biology and how they evolve. For example, genetic sequencing techniques have been used to identify mutations associated with drug resistance. These links can provide valuable information to aid in the design of new cancer-fighting strategies.
Moreover, there is a need to understand the interaction between cancer tissues and the immune system. Focusing on the use of immunotherapies – such as T-cell therapy and targeted drugs – has proven successful in some types of cancer, but broader applications are still under development. New ideas in research and clinical studies must converge to enhance the effectiveness of these therapeutic strategies.
Clinical Applications of Stem Cells
Stem cells are among the most prominent topics in regenerative medicine, offering great potential for treating a variety of diseases. They are used in developing new therapies, particularly for issues related to damaged tissues or chronic diseases. As the use of stem cell culturing techniques increases, it is important to evaluate the risks associated with using these cells to ensure patient safety.
Research shows that stem cells can contribute to tissue regeneration and reduce damage from injuries. For example, the feasibility of using stem cells derived from umbilical cord blood for treating autoimmune diseases has been studied. Notably, these cells represent an almost unlimited source of stem cells that can be used for therapy.
However, despite these benefits, there are risks related to carcinogenesis, as stem cells may acquire cancerous properties if their culture is not monitored properly. This situation requires the development of precise strategies to mitigate these risks, calling for the need for advanced analysis and screening processes prior to moving on to clinical trials.
New Systems for Genome Analysis
With the advancement of genome analysis technology, the possibilities for understanding the genetic factors associated with diseases are increasing. New techniques allow scientists to analyze data quickly and accurately, facilitating the study of genetic variations that may contribute to the development of a specific disease. Accurate analyses of genomic data also help in identifying potential treatment responses, thereby improving therapeutic outcomes.
An obvious example is how modern technologies are used to identify genetic mutations associated with drug resistance in cancer patients. By analyzing the genome, researchers can monitor genetic changes that may affect how cells respond to treatment.
This leads to…
The use of these new systems aims to improve diagnostics and predictions regarding patients, enabling doctors to tailor treatments based on the individual’s genetic analysis. This personalized approach holds great potential for enhancing treatment outcomes and increasing the effectiveness of drugs used for therapeutic purposes. However, it remains essential to address the ethical challenges and policies associated with the widespread use of these technologies to ensure patient safety and privacy.
Source link: https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2024.1388312/full
Artificial intelligence was used ezycontent
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