A complete flow cytometric analysis includes: sample preparation, flow cytometry, and data analysis. The importance of cell sample preparation in the flow analysis process is undoubted, directly affecting the accuracy of the data and the overall progress of the experiment. However, during the preparation of traditional cell samples, centrifugation may cause many defects to the cells , such as: crushing and destroying cells; uncontrollable cell loss causes the cell population in the original sample to deviate, resulting in severe distortion of the data, allowing research It is possible to obtain non-objective data analysis and diagnostic results. In addition, traditional centrifugation is also significantly affected by operator variability and requires a lot of manpower and material resources.
Therefore, in order to obtain more scientific experimental data, it is the key to improve the cell preparation process by entering a new realm of flow cytometric analysis that can be quantitative and reproducible.
The DA-Cell microdrop array sample pretreatment system, as a new generation of sample pretreatment system, utilizes the effects of gravity and laminar flow to achieve suspended samples of cells, beads, etc. in a very light and gentle scientific manner without centrifugation. Cleaning operation. Its advantages in the cell preparation process are unmatched by traditional centrifugation methods.
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Adicet Bio , a company specializing in the development of cellular immunotherapy, signed a five- year partnership with Regeneron Pharmaceuticals in 2016 to collaborate on multiple projects including solid tumors and hematologic malignancies ( including Allogeneic cell therapy ) . In order to accurately engage and kill tumor cells, Adicet Bio is developing an engineered chimeric antigen receptor (CAR) and T cell receptor (TCR) based on γδ T cells to specifically target intracellular and extracellular antigens. Hematological and solid tumors offer a diverse range of clinical candidates.
The in vitro preparation process of γδT cells (ICP) products includes the following steps:
1. Isolation and extraction of γδT cells from healthy donors
2. Genetic modification of γδT cells
3. γδT cell expansion
4. The amplified γδT cell product is deposited in the cell bank for subsequent use.
Adicet Bio uses a flow cytometry-based sorting protocol at the 2nd, 3rd, and 4th steps to sample and control different donors to ensure that subsequent products for treatment meet the requirements. The content of γδ T in human blood is very low. If the donor's cell loss is severe during sample preparation, the researchers may misjudge the result. To avoid this and get more realistic and scientific streaming data to select the most appropriate sample, Adicet Bio researchers used the DA-Cell microdrop array sample before the cell sample preparation process for the sorting protocol. Processing system.
Result 1: DA-Cell can improve streaming data quality
High-quality flow cytometry data can help researchers properly sort donor samples and quality control, and reduce false positives due to false positive results.
Result 2: DA-Cell makes the experiment highly reproducible
DA-Cell has a high degree of automation, avoiding the error of manual operation, greatly improving the repeatability of the experiment, and even if the sample is rare, it can obtain high repeatability.
Result 3:: DA-Cell has a high degree of data stability
Cell samples were processed several times by DA-Cell, and the proportion of cell subpopulations remained the same, maximally avoiding data bias.
Result 4: DA-Cell saves a lot of time
DA-Cell is highly automated and requires only 10-20% of the time of traditional centrifugation methods to increase experimental efficiency.
to sum up
The DA-Cell microdrop array sample pretreatment system subverts the traditional centrifugation method. The principle is to use the laminar flow effect of gravity and fluid mechanics to achieve cells and beads in a very light and gentle scientific way in the case of non-centrifugation. The cleaning operation of the suspended sample. Compared to traditional centrifugal methods, DA-Cell offers great advantages:
1. Eliminate data deviation caused by traditional centrifugation and obtain more scientific results
Because the size, density, and morphology of different cells are different, they lose different proportions in the traditional centrifugation process. The retained ratio has deviated from the original true value, which may lead to deviations or even errors in the results. The DA-Cell method has minimal cell loss, which preserves the original cell ratio to the greatest extent and helps to obtain more realistic and scientific results.
â–³ conventional leaving only 20-30% of the cells from samples Center Weighted method, DA-Cell samples can be retained more than 95% of the cells
â–³ compared to traditional off Center Weighted shutter mode, DA-Cell may exhibit a stronger fluorescence signal with intact cells morphology
â–³ â½£ extrusion processing will seriously affect cell function caused by expression of egg white from Center Weighted process
2. Save time, improve efficiency and throughput
Comparison of traditional centrifugation and DA-Cell method for preparing cell samples
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Conventional centrifugation has multiple steps for each cleaning, including: adding liquid, resuspension, centrifugation, de-clearing, etc. If it is washed 2-4 times, it takes 20-40 minutes, while the DA-Cell method only requires 2- In 6 minutes, only 10-20% of the time is required for the traditional method .
3. The sample processing process is very gentle, to prevent stress and cell damage Â
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The preparation of suspended samples such as traditional cells and microbeads must be carried out by centrifugation, and the centrifugal force reaches hundreds of g or thousands of g (that is, the cells need to withstand hundreds or thousands of times their own weight), which affects the state of the cells. It is extremely large and even broken. DA-Cell can only use the gravity and hydrodynamic laminar effects of the cell to achieve the preparation process of the sample without any stress on the cells, so the cell damage can be significantly reduced.
â–³ Comparative PBMC samples after processing DA-Cell 4X treated vs 4X centrifugal effect on cell viability of
â–³ DA-Cell is significantly higher than the traditional centrifugation method in the ratio of living cells
â–³ data show that traditional centrifugation is more likely to cause more cell damage and even cell death than DA-Cell
4. Reduce cell stress, maintain the most primitive protein and biomarker expression levels
Reducing cell stress, maintaining the most primitive protein and biomarker expression levels Traditionally, centrifugation can stress a large number of suspended sample products, which affects the expression levels of many genes and ultimately affects the proteins and organisms that scientists are observing. Markers, resulting in biased data. The DA-Cell microdrop array sample preparation system has minimal stress and interference on cells, minimizing interference with protein and biomarker expression levels.
â–³ The positive value indicates that the dyeing index is higher. After using DA-Cell, the protein staining degree is relatively increased. â–³ Effective observation of proteins and biomarkers, the experimental results are more intuitive.
5. Improve streaming analysis data results
DA-Cel can clean cells in a very gentle and scientific way without centrifugation, and the obtained data can easily obtain reliable cell clustering results even with multi-color flow analysis.
Comparison of samples in 16- color fluorescence flow analysis after traditional centrifugation and DA-Cell treatment
â–³ HSC / iNKT: DA-Cell because they can reduce cell loss and signal that allows the experimenter to see the rare cell subsets, free from the traditional Center Weighted law
â–³ GDT / MAIT: DA-Cell significantly improve the boundary between the cell subpopulations, on the conventional method from Center Weighted still pictures easily broken due to excessive cell cause difficulties distinguishing
6. Fully automatic operation, better repeatability, avoiding deviations from manual introduction
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The traditional centrifugal method has many artificial factors. Because different people's operation methods are different, and the same person's operation methods at different times are difficult to maintain consistent, the experiment is less repeatable; and DA-Cell is fully automatic operation, avoiding manual introduction. The error can greatly improve the repeatability of the experiment.
The devil is hidden in the details, the details determine the success or failure, DA-Cell can improve the quality of the data in the details of cell processing, and make the flow cell analysis into a new realm of quantitative and reproducible, thus improving the precise monitoring of cellular immunotherapy. ,
There are also some raw materials, orlistat, liraglutide, cetilistat, etc.
Therefore, in order to obtain more scientific experimental data, it is the key to improve the cell preparation process by entering a new realm of flow cytometric analysis that can be quantitative and reproducible.
The DA-Cell microdrop array sample pretreatment system, as a new generation of sample pretreatment system, utilizes the effects of gravity and laminar flow to achieve suspended samples of cells, beads, etc. in a very light and gentle scientific manner without centrifugation. Cleaning operation. Its advantages in the cell preparation process are unmatched by traditional centrifugation methods.
         Â
Adicet Bio , a company specializing in the development of cellular immunotherapy, signed a five- year partnership with Regeneron Pharmaceuticals in 2016 to collaborate on multiple projects including solid tumors and hematologic malignancies ( including Allogeneic cell therapy ) . In order to accurately engage and kill tumor cells, Adicet Bio is developing an engineered chimeric antigen receptor (CAR) and T cell receptor (TCR) based on γδ T cells to specifically target intracellular and extracellular antigens. Hematological and solid tumors offer a diverse range of clinical candidates.
The in vitro preparation process of γδT cells (ICP) products includes the following steps:
1. Isolation and extraction of γδT cells from healthy donors
2. Genetic modification of γδT cells
3. γδT cell expansion
4. The amplified γδT cell product is deposited in the cell bank for subsequent use.
Figure 1. In vitro preparation of γδT cell products
Adicet Bio uses a flow cytometry-based sorting protocol at the 2nd, 3rd, and 4th steps to sample and control different donors to ensure that subsequent products for treatment meet the requirements. The content of γδ T in human blood is very low. If the donor's cell loss is severe during sample preparation, the researchers may misjudge the result. To avoid this and get more realistic and scientific streaming data to select the most appropriate sample, Adicet Bio researchers used the DA-Cell microdrop array sample before the cell sample preparation process for the sorting protocol. Processing system.
Figure 2. The donor's sample contains different proportions of γδT cells.
Figure 3. Sample sorting scheme based on flow cytometry
Result 1: DA-Cell can improve streaming data quality
High-quality flow cytometry data can help researchers properly sort donor samples and quality control, and reduce false positives due to false positive results.
Result 2: DA-Cell makes the experiment highly reproducible
DA-Cell has a high degree of automation, avoiding the error of manual operation, greatly improving the repeatability of the experiment, and even if the sample is rare, it can obtain high repeatability.
Result 3:: DA-Cell has a high degree of data stability
Cell samples were processed several times by DA-Cell, and the proportion of cell subpopulations remained the same, maximally avoiding data bias.
Result 4: DA-Cell saves a lot of time
DA-Cell is highly automated and requires only 10-20% of the time of traditional centrifugation methods to increase experimental efficiency.
to sum up
The DA-Cell microdrop array sample pretreatment system subverts the traditional centrifugation method. The principle is to use the laminar flow effect of gravity and fluid mechanics to achieve cells and beads in a very light and gentle scientific way in the case of non-centrifugation. The cleaning operation of the suspended sample. Compared to traditional centrifugal methods, DA-Cell offers great advantages:
1. Eliminate data deviation caused by traditional centrifugation and obtain more scientific results
Because the size, density, and morphology of different cells are different, they lose different proportions in the traditional centrifugation process. The retained ratio has deviated from the original true value, which may lead to deviations or even errors in the results. The DA-Cell method has minimal cell loss, which preserves the original cell ratio to the greatest extent and helps to obtain more realistic and scientific results.
â–³ conventional leaving only 20-30% of the cells from samples Center Weighted method, DA-Cell samples can be retained more than 95% of the cells
â–³ compared to traditional off Center Weighted shutter mode, DA-Cell may exhibit a stronger fluorescence signal with intact cells morphology
â–³ â½£ extrusion processing will seriously affect cell function caused by expression of egg white from Center Weighted process
2. Save time, improve efficiency and throughput
Comparison of traditional centrifugation and DA-Cell method for preparing cell samples
Â
Conventional centrifugation has multiple steps for each cleaning, including: adding liquid, resuspension, centrifugation, de-clearing, etc. If it is washed 2-4 times, it takes 20-40 minutes, while the DA-Cell method only requires 2- In 6 minutes, only 10-20% of the time is required for the traditional method .
3. The sample processing process is very gentle, to prevent stress and cell damage Â
Â
The preparation of suspended samples such as traditional cells and microbeads must be carried out by centrifugation, and the centrifugal force reaches hundreds of g or thousands of g (that is, the cells need to withstand hundreds or thousands of times their own weight), which affects the state of the cells. It is extremely large and even broken. DA-Cell can only use the gravity and hydrodynamic laminar effects of the cell to achieve the preparation process of the sample without any stress on the cells, so the cell damage can be significantly reduced.
â–³ Comparative PBMC samples after processing DA-Cell 4X treated vs 4X centrifugal effect on cell viability of
â–³ DA-Cell is significantly higher than the traditional centrifugation method in the ratio of living cells
â–³ data show that traditional centrifugation is more likely to cause more cell damage and even cell death than DA-Cell
4. Reduce cell stress, maintain the most primitive protein and biomarker expression levels
Reducing cell stress, maintaining the most primitive protein and biomarker expression levels Traditionally, centrifugation can stress a large number of suspended sample products, which affects the expression levels of many genes and ultimately affects the proteins and organisms that scientists are observing. Markers, resulting in biased data. The DA-Cell microdrop array sample preparation system has minimal stress and interference on cells, minimizing interference with protein and biomarker expression levels.
â–³ The positive value indicates that the dyeing index is higher. After using DA-Cell, the protein staining degree is relatively increased. â–³ Effective observation of proteins and biomarkers, the experimental results are more intuitive.
5. Improve streaming analysis data results
DA-Cel can clean cells in a very gentle and scientific way without centrifugation, and the obtained data can easily obtain reliable cell clustering results even with multi-color flow analysis.
Comparison of samples in 16- color fluorescence flow analysis after traditional centrifugation and DA-Cell treatment
â–³ HSC / iNKT: DA-Cell because they can reduce cell loss and signal that allows the experimenter to see the rare cell subsets, free from the traditional Center Weighted law
â–³ GDT / MAIT: DA-Cell significantly improve the boundary between the cell subpopulations, on the conventional method from Center Weighted still pictures easily broken due to excessive cell cause difficulties distinguishing
6. Fully automatic operation, better repeatability, avoiding deviations from manual introduction
Â
The traditional centrifugal method has many artificial factors. Because different people's operation methods are different, and the same person's operation methods at different times are difficult to maintain consistent, the experiment is less repeatable; and DA-Cell is fully automatic operation, avoiding manual introduction. The error can greatly improve the repeatability of the experiment.
The devil is hidden in the details, the details determine the success or failure, DA-Cell can improve the quality of the data in the details of cell processing, and make the flow cell analysis into a new realm of quantitative and reproducible, thus improving the precise monitoring of cellular immunotherapy. ,
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