CN112680529B - Quality control method of C3A cells required by biotype artificial liver treatment and application thereof - Google Patents
Quality control method of C3A cells required by biotype artificial liver treatment and application thereof Download PDFInfo
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Abstract
The invention discloses a quality control method of C3A cells required by biotype artificial liver treatment and application thereof. The C3A cells obtained by screening by the quality control method have good quality and high safety, and can be used for large-scale production and treatment of the biological artificial liver.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to a quality control method of C3A cells required by biotype artificial liver treatment and application thereof.
Background
The liver is a complex organ composed of several different cell types, but the key is the hepatocyte, which plays most of its biochemical functions in the process of acute phase reaction, coagulation factor production and biochemical reaction for detoxification. When hepatocytes are cultured or expanded in vitro, their viability is limited because they quickly stop differentiating or die. Thus, liver assist products present practical and theoretical hurdles in using normal hepatocytes. Cell lines derived from well-differentiated liver tumor cells alleviate many of the practical and theoretical hurdles. The specific cell line was selected for the human allogeneic ELAD (extracorpuscular river Assist device), i.e. the human C3A cell line, which is a subclone of the human hepatoblastoma cell line HepG 2. C3A cells proliferate rapidly immortalize and retain the same liver function to facilitate the mass of cultured growing cells needed for subjects treated as a liver support system to perform impaired liver function and maintain cell proliferation, differentiation and metabolic activity during treatment.
Mutation, senescence and evolution occur continuously in all actively growing cell cultures. The longer the maintenance and amplification time, the more these changes accumulate. Thus, cell cultures often lose important genotypic and/or phenotypic characteristics, such as alterations in biochemical and functional properties. Cell lines derived from tumors or genetic engineering are particularly susceptible to genetic alterations. In addition to the effect on cell line characteristics, the use of an overcontinuous cell line increases cell stability, the risk of cross contamination of microorganisms, viruses or cells. A key step in controlling these problems and achieving "sourcing" or standardization of cell lines is the establishment and careful maintenance of frozen cell stocks for all key cell lines. A complete cell bank is crucial to ensure that there is an adequate equivalent supply of cells throughout the life of the product production, and to maintain product quality and industrialization. The purpose of cell bank characterization is to determine the character, purity and suitability of the cell matrix for production use. Careful testing is required for thawed cells to verify the following: 1) Activity; 2) Free of mycoplasma or other microbial contaminants; 3) Cell line identity; 4) Important cell line characteristics to ensure stability of cell properties and safety of cell use.
Quality control of C3A cells required for ELAD is very important because only a sufficient high quality C3A cell stock can meet the needs of a wide range of clinical liver disease patients, promote recovery of liver cell regeneration function of the patients themselves, or prolong the life span of the patients.
Disclosure of Invention
One of the purposes of the invention is to provide a quality control method of C3A cells required by biotype artificial liver treatment.
The second objective of the present invention is to provide a cell bank of human C3A cell line, which ensures large-scale production of the biological artificial liver.
It is a further object of the present invention to provide the use of the above quality control method.
In order to achieve the purpose, the invention adopts the following technical scheme:
according to one aspect of the invention, the invention provides a quality control method of C3A cells required by treatment of a biotype artificial liver, which ensures the treatment quality of the biotype artificial liver.
Further, the quality control method comprises detecting cell property stability and cell safety;
in a specific embodiment of the present invention, the quality control method comprises the steps of:
1) Establishing a main cell bank, detecting the property stability of the cells and detecting the safety of the cells;
2) Establishing a manufacturer working cell bank, detecting the property stability of the cells and the safety of the cells;
3) Establishing a working cell bank, detecting the property stability of the cells and detecting the safety of the cells;
4) Establishing a cell bank after production is finished, detecting the property stability of the cells and detecting the safety of the cells;
further, detecting the stability of the cell property includes detecting the genotype, phenotype and activity of the cell.
In a specific embodiment of the invention, the detection of cell genotype uses a short tandem repeat test.
Genotypes for use in the short tandem repeat test of the invention include AMEL, D3S1358, D1S1656, D6S1043, D13S317, penta E, D16S539, D18S51, D2S1338, CSF1PO, penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA.
Further, detecting cell safety includes detecting whether microbial contamination exists in the cells, detecting whether viral contamination exists in the cells, detecting whether cross contamination exists between cells, and detecting whether microbial contamination exists in a device for culturing the cells.
Further, the microorganism includes bacteria, fungi, yeast, mycoplasma.
Further, viruses include endogenous viruses and exogenous viruses.
Still further, the virus includes human virus, non-human virus. Preferably, the non-human virus comprises murine virus, porcine virus, bovine virus.
In a particular embodiment of the invention, the human virus comprises HSV-1/2, B19, EBV, SV40, HHV5/CMV, HHV6, HHV7, HHV8, HIV-1, HIV-2, HBV, HCV, HTLV, HAV, HEV.
In a specific embodiment of the invention, the murine virus includes murine pox virus, rat taylor encephalomyelitis virus, lactate dehydrogenase virus, lymphocytic choriomeningitis virus, hantavirus, mouse parvovirus, mouse adenovirus, mouse hepatitis virus, mouse pneumovirus, polyoma virus, sendai virus, juvenile epidemic diarrhea virus, mouse salivary adenovirus (mouse cytomegalovirus), reovirus type 3, virus K, mouse thymic adenovirus, mouse thermophilic retrovirus, mouse retrovirus.
In a particular embodiment of the invention, the porcine virus comprises porcine parvovirus.
Methods for detecting cells and fungi that can be used in the present invention include direct inoculation, membrane filtration.
Methods for detecting mycoplasma that can be used in the present invention include culture assays, non-culture assays.
Further, non-culture assays include microscopy, nucleic acid assays.
Furthermore, the nucleic acid detection comprises a PCR method, a nested PCR method, a fluorescent quantitative PCR method, reverse dot hybridization, surface plasmon resonance biosensor rapid detection, a fluorescence polarization technology and the like.
Methods for detecting viruses that can be used in the present invention include thin layer electron microscopy, infectivity assays, cell co-culture, PCR, PERT, extended XC plaque, extended mink S + L.
In a specific embodiment of the invention, the method of creating a master cell library comprises the steps of: the C3A cells were sub-cultured and expanded at 3X 10 in a cell culture facility 6 To 1X 10 7 The cell density of the cells/bottles is frozen in a frozen preservation solution; preferably, the cryopreservation solution comprises 10% DMSO, 20% fetal bovine serum, MM medium (available from Shandong Yi Ruite Biotech Co., ltd.).
In a particular embodiment of the present invention,the method for establishing the manufacturer working cell bank comprises the following steps: recovering, culturing and expanding cells in the main cell bank, passaging the cells for 4 times, and averaging the 5 th generation cells to 1.08 × 10 7 The cell density of each cell/bottle is frozen and stored in a frozen and stored solution; preferably, the cryopreservation solution comprises 10% glycerol, 5% fetal bovine serum and MM culture solution.
In a particular embodiment of the invention, the method of establishing a working cell bank comprises the steps of: cells in the manufacturer's working cell bank were recovered, cultured, and expanded by about 10 7 The cell density of each cell/bottle is frozen and stored in a frozen and stored solution; preferably, the cryopreservation solution comprises 20% fetal bovine serum, 10% DMSO, MM medium.
In a specific embodiment of the present invention, the method for establishing the production-terminated cell bank comprises the steps of recovering, culturing and passaging the cells in the working cell bank in a culture flask for 7 times, and simulating the whole process of culturing and growing the cells in a bioreactor.
According to another aspect of the invention, there is provided the use of the method described above for the construction of a C3A cell bank.
Further, the cell bank comprises a main cell bank, a manufacturer working cell bank, a working cell bank and a production end cell bank.
The definition of the master cell bank, the manufacturer's working cell bank, the working cell bank and the end-of-production cell bank is the same as that described above.
According to another aspect of the invention, there is provided the use of a method as hereinbefore described in the preparation of C3A cells for use in the treatment of an artificial liver assist device.
The term "master cell bank" as used herein refers to a batch of primary cell seeds which are uniformly mixed after subculture and proliferation, quantitatively packaged, and stored in liquid nitrogen or below-130 ℃. After the cells are completely qualified, the cells are taken as a main cell bank for establishing a working cell bank.
As used herein, the term "manufacturer's working cell bank" refers to a large number of uniformly composed cells obtained from the seeds of cells in a master cell bank, stored in aliquots at a temperature of-130 ℃ or less, for production. In normal practice, cell seed expansion is selected by the manufacturer when subcultured to a certain passage number. The cells are merged into a pool, and the cells are equally divided into bottles to be frozen and stored to obtain the MWCB.
The working cell bank refers to cells which are subjected to subculture proliferation by a manufacturer to reach a certain generation level, are mixed to prepare a batch of homogeneous cell suspension, are quantitatively packaged in a certain number of cell cryopreservation tubes, and are stored in liquid nitrogen or at the temperature of below 130 ℃ below zero for later use. And the cells are qualified through complete verification, and then the cells are the working cell bank.
As used herein, the term "end-of-production cell bank" refers to a cell bank comprised of cells harvested at or beyond the end of production. The method is used for detecting the characteristics and the existence of changes of molecular structure level after cells are cultured for a long time to become products.
As used herein, "bioartificial liver (BAL)" refers generally to an in vitro cell bioreactor device constructed based on artificially cultured hepatocytes.
The cell bioreactor is the core of the entire BAL. The cell bioreactors currently studied and applied are mainly the following:
1) Hollow fiber type cell bioreactor
Is the most applied type of reactor at present. The hollow fiber tube is divided into an inner cavity and an outer cavity, the fiber membrane is a layer of barrier, the liver cells are generally cultured and grown in the outer cavity at present, the cells grow in a three-dimensional mode, and the membrane can prevent the cells from entering a human body. The reactor has the greatest advantages of huge surface area to volume ratio, convenience for metabolite transportation and minimal dead space. However, the activity and function of the cells are also easily reduced due to uneven distribution of the hepatocytes in the reactor.
2) Flat plate single layer bioreactor
The reactor is used for directly planting the liver cells on a flat plate, and has the advantages that the cells are uniformly distributed, the microenvironment is consistent, but the ratio of the surface area to the volume is reduced, and Shito et al designs a flat plate monolayer bioreactor with an inner membrane oxygenator.
3) Perfusion bed/stent bioreactor
The bioreactor is used for planting the liver cells on an infusion bed or a bracket, the reactor enables the plasma to be directly contacted with the cells, the transfer of substances is increased, the three-dimensional bracket also promotes the formation of the three-dimensional structure of the liver cells, the cell capacity is easily expanded, and the bioreactor has the defects of nonuniform infusion, easy blockage and easy entry of variant cells into a human body.
4) Coating suspension bioreactor
The bioreactor is prepared by wrapping liver cells with a semipermeable membrane material to prepare porous microcapsules, and then performing perfusion culture. Its advantages are the same microenvironment for all cells, and large cell culture space, resulting in less immunoreaction. The disadvantages are poor cell stability, easy clogging of the micropores, limited material exchange capacity and limited cell quantity obtained.
One type of biotype artificial liver commonly used is the in vitro liver assist device (ELAD) designed by scientists at Baylar medical college, USA, vitamin therapeutics, inc (San Diego, CA, USA) developed further and conceptually similar to an assisted liver, which uses C3A hepatocytes rather than animal hepatocytes, including four hollow fiber dialysis reactors. Before use, a small number of hepatocytes were seeded into each reactor. After 2-3wk of maturation period. The cells are replicated and amplified. Each reactor contained approximately 100-120g of hepatocytes. Ultrafiltration (150-200 mL/min) was achieved by a venous-venous hemofilter. A significant advantage of this filter design is that it can be operated continuously for 10 days without interruption. The ultrafiltration membrane only allows substances with the diameter of 100-120KD to pass through, and is also called ultrafiltrate
The invention has the advantages and beneficial effects that:
the invention discloses the quality control that the C3A cells required by the preparation of biotype artificial liver treatment must carry out for the first time. Only through the quality control, the C3A cell with stable cell property and strong safety can be obtained.
The invention constructs a C3A cell bank for the first time, and the cell bank is divided into a main cell bank, a manufacturer working cell bank, a working cell bank and a production finishing cell bank according to different purposes. Enough cell freezing bottles are stored in the cell bank, high-quality fresh cultured cells can be continuously provided according to the requirement, and the large-scale mass production of the high-quality biological cell reactor can be ensured.
Drawings
FIG. 1 shows a flow chart for the construction of a final cell bank.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure. In the following examples, the reagents used were all analytical grade and were commercially available unless otherwise indicated. The experimental procedures not specified for the specific conditions are generally carried out under conventional conditions such as those described in the book "molecular cloning Experimental Manual", published 2002 by scientific Press, J. SammBruker et al, or under conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.
Example preparation, manufacture, characterization of cell banks and safety testing of adventitious extrinsic factors
1. Master cell library construction
C3A cells are cultured and maintained in a cell culture facility. C3A cells were sub-cultured and expanded until there were enough cells to be 3X 10 6 To 1X 10 7 Cells/flasks were cryopreserved. Collecting cells at logarithmic growth phase, using
NC-3000 TM An advanced image cytometer uses fluorescence imaging to characterize cell characteristics and viable cell viability indices for cell counting. After centrifugation to remove the culture supernatant, the cells were resuspended at the desired concentration in a cryopreservative containing 10% dimethylsulfoxide and 20% fetal bovine serum. The resuspended cells were dispersed in cryovials. 140 vials containing cells of the Master Cell Bank (MCB, master Cell Bank) were prepared, each vial containing 3X 10 cells 6 To 1X 10 7 (average 5.07X 10) 6 ) And (4) living cells. The cryopreservation solution containing 10% dimethyl sulfoxide and 20% fetal bovine serum refers to MM culture solution (available from Shandong Yi Ruite Biotech Co., ltd.) containing 10% DMSO and 20% fetal bovine serum.
Isoenzyme analysis was also performed to confirm that these cells were of human origin. Table 1 lists the results of testing MCB under GLP conditions. These results demonstrate the identity and genetic stability of C3A cells in MCB. It was also confirmed that MCB tested did not contain endogenous or exogenous viruses, retroviruses and other endogenous viruses. Note: in table 1 "unobvious virus" is recessive infection, subclinical infection; infection without clinical symptoms. Lymphocytic choriomeningitis virus (LCMV) is a rodent transmitted sandtype virus that causes insignificant infection).
TABLE 1 detection results of MCB cell characteristics
2. Production Manufacturer's Working Cell Bank (MWCB) construction
Cell expansion was initiated from C3A in a vial of MCB. Cells were passaged four times and stored frozen at passage 5 in MM culturesIn the medium, MM medium containing 10% glycerol and 5% fetal bovine serum was owned by Shandong Yiruite Biotech Co. In total 107 vials were produced, each vial containing on average 1.08X 10 7 The average survival rate of each cell reaches 78 percent.
Table 2 lists the extrinsic factor (e.g., virus and mycoplasma) tests performed on C3A MWCB. The test results confirm that the identity of the MWCB is preserved and maintained.
TABLE 2 detection results of MWCB cell characteristics
| Description of the detection | As a result, the | |
| 1 | Sterility testing by direct inoculation (bacteria and fungi) | No growth |
| 2 | Mycoplasma (PTC) | Negative of |
| 3 | In vitro determination of foreign viral contaminants (protocol # 003031) | No detection out |
| 4 | In vitro assay for foreign viral contaminants (protocol # 005002) | No detection out |
| 5 | Analytical identification and characterization of cell cultures using the isozyme method | Human origin |
| 6 | Identity recognition by short tandem repeat analysis | 100% identity |
3. Construction of Working Cell Bank (Working Cell Bank, WCB)
Two MWCB vials were used to produce the C3A Working Cell Bank (WCB). Using a protocol and procedure similar to MWCB production to produce 200 WCB vials, cells were suspended in 20% fetal bovine serum (BCS), 10% DMSO and cryo-medium equilibrated with MM medium, averaging about 107 cells per vial. The tests performed on the final WCB vials are shown in table 3. The four media in table 3 refer to: 1) Soy casein digestion broth (TSB); 2) Thioglycolic acid liquid broth (THIO); 3) Saxifrage glucose agar; 4) Sterile Phosphate Buffered Saline (PBS) was used as a negative control for inoculation.
TABLE 3 WCB cell characterization assay
4. Production of End cell Bank (End of Production Cells Bank, EOPCB)
A study at the end of production was initiated using a standard C3A cell culture expansion procedure. Cells were passaged 8 times within 8 weeks, estimated to correspond to about 16-24 cell doublings. Once the C3A cells were seeded into the ELAD C3A cell reactor, it was estimated that the reactor would produce an effect equivalent to 7 additional passages. However, since cells cannot be recovered after introduction into the cell reactor, the study in this section simulated cell growth/production in the ELAD C3A cell reactor using T225 culture flasks. These cells were subjected to an additional 7 passages over a period of 7 weeks in T225 flasks, estimated to correspond to 14-21 additional doublings. Based on this design, the cells tested at the end of the production study were 15 weeks old, undergoing a total of about 15 passages, corresponding to approximately 30-45 doublings. During the course of this study, cells were exposed to fresh medium (containing bovine serum) approximately 45 times. A flow chart for the preparation of the final cell bank is shown in FIG. 1.
Viral safety and characterization tests were performed at the end of the in vitro cell age limit and showed that the cells retained their identity as C3A cells at the end of production and were free of viral contaminants. The test results are provided in table 4.
The 14 viruses (Human specific Type I) in Table 4 are HSV-1/2 (Human Simplex Virus Types 1 and 2, herpes Simplex viruses 1 and 2), B19 (parvovirus B19), EBV (Epstein-Barr Virus ), SV40 (Simian Virus 40, simian Virus Type 40), HHV5/CMV (Human Herpesvirus Type 5/Human Cytomegalovirus ), HHV6 (Human Herpesvirus Type 6, human Herpes Virus Type 6), HHV7 (Human Herpesvirus Type 7 ), HHV8 (Human Herpesvirus Type 8, human Herpes Virus Type 8), HIV-1 (Human Herpesvirus Type 1, human Immunodeficiency Virus Type 2, human Immunodeficiency Virus Type 2 (Human Immunodeficiency Virus Type 2, hepatitis B), human Immunodeficiency Virus Type 2 (Hepatitis B, hepatitis C), hepatitis B Virus Type 2, hepatitis C (Hepatitis B), hepatitis C Virus Type 2, hepatitis B), hepatitis B Virus Type Hepatitis C (Hepatitis C).
The 17 murine viruses in Table 4 are murine pox Virus (Ectromeal Virus), rat Taylor encephalomyelitis Virus (GDVII), lactate Dehydrogenase Virus (Lactate Dehydrogenase Virus (LDV)), lymphocytic Choriomeningitis Virus (Lymphocytic meningitis Virus), hantaan Virus (Hantaan Virus), mouse Parvovirus (Mouse Minute Virus (MMV)), mouse Parvovirus (Mouse Parvovirus (MPV)), mouse Adenovirus (Mouse Adenovirus) Mouse Hepatitis Virus (MHV), mouse Pneumonia Virus (Pneumonia Virus of Rice (PVM)), polyoma Virus (Polyoma Virus), sendai Virus (Sendai Virus), juvenile epidemic Diarrhea Virus (epidemic Diarra of Infant Rice (EDIM)), mouse Salivary Adenovirus (Mouse Cytomegalovirus) (Mouse Salivary and Virus (MCMV)), reovirus Type 3 (Reovirus Type 3), K Virus, mouse Thymic Adenovirus (MTV)).
TABLE 4 EPCB (End of Production Cells Bank) Production End cell characterization
As part of the cell characterization test, a Short Tandem Repeat (STR) test was used to confirm the genotype of the C3A cell bank. The results are shown in table 5, showing that all cell banks retain their identity and characteristics as C3A cells without any variation and change.
TABLE 5 C3A cell bank STR assay
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Claims (3)
1. A quality control method of C3A cells required by biotype artificial liver treatment is characterized by comprising the steps of detecting the property stability of the cells and detecting the safety of the cells;
the quality control method comprises the following steps:
1) Establishing a main cell bank, detecting the property stability of the cells and detecting the safety of the cells;
2) Establishing a manufacturer working cell bank, detecting the property stability of the cells and the safety of the cells;
3) Establishing a working cell bank, detecting the property stability of the cells and detecting the safety of the cells;
4) Establishing a production terminal cell bank, detecting the property stability of the cells and detecting the safety of the cells;
the method for establishing the master cell library comprises the following steps: the C3A cells were sub-cultured and expanded at 3X 10 in a cell culture facility 6 To 1 × 10 7 The cell density of the cells/bottles is frozen in a frozen preservation solution; the cryopreservation solution comprises 10% DMSO, 20% fetal calf serum and MM culture solution;
the method for establishing the manufacturer working cell bank comprises the following steps: recovering, culturing and amplifying cells in the main cell bank, passaging the cells for 4 times, and culturing the cells of the 5 th generation at a ratio of 1.08 × 10 7 The cell density of each cell/bottle is frozen in a frozen preservation solution; the cryopreservation liquid comprises 10% of glycerol, 5% of fetal calf serum and MM culture solution;
the method for establishing the working cell bank comprises the following steps: cells in the manufacturer's working cell bank were thawed, cultured, expanded at 10 7 The cell density of each cell/bottle is frozen and stored in a frozen and stored solution; the cryopreservation solution comprises 20% fetal calf serum, 10% DMSO and MM culture solution;
the method for establishing the final cell bank comprises the steps of reviving, culturing and passaging cells in a working cell bank in a culture flask for 7 times;
detecting the stability of cell properties comprises detecting the genotype, phenotype and activity of the cell;
detection of cell genotype using short tandem repeat testing; the short tandem repeat sequence includes AMEL, D3S1358, D1S1656, D6S1043, D13S317, penta E, D16S539, D18S51, D2S1338, CSF1PO, penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA.
2. The method of claim 1, wherein detecting cell safety comprises detecting the presence of bacterial, fungal, mycoplasma contamination of the cells, detecting the presence of viral contamination of the cells, detecting the presence of cell cross contamination of the cells, detecting the presence of contamination of a device in which the cells are cultured; the virus comprises HSV-1/2, B19, EBV, SV40, HHV5/CMV, HHV6, HHV7, HHV8, HIV-1, HIV-2, HBV, HCV, HTLV, HAV, HEV, mousepox virus, rat taylor encephalomyelitis virus, lactate dehydrogenase virus, lymphocytic choriomeningitis virus, hantaan virus, mouse parvovirus, mouse adenovirus, mouse hepatitis virus, mouse pneumovirus, polyoma virus, sendai virus, juvenile epidemic diarrhea virus, mouse salivary adenovirus, reovirus type 3, K virus, mouse thymic virus, mouse retrovirus and porcine parvovirus.
3. Use of the method of claim 1 or 2 for the preparation of C3A cells for biotype artificial liver therapy.
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