CN110106142B - Production process for preparing billion-grade fat source regenerative cells - Google Patents
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Abstract
The invention belongs to the field of regenerative medicine biotechnology, and particularly relates to an amplification culture method of billion-grade adipose-derived regenerative cells. The invention inoculates and attaches the adipose-derived regenerative cells to a fixed packed bed arranged by a sheet-shaped carrier in a cell culture container by a 'batch inoculation' process of the cells, and improves the billion level of the prior art to the billion level of cell amplification by combining a 'fed-batch perfusion' process. The invention abandons the use of disposable cell culture containers and a large amount of expensive collagen microcarriers, greatly reduces the fixed cost accounting for the maximum of the product sale cost, and is a remarkable progress of the invention. The cell culture vessels used in the present invention and the closest prior art are newer iterative models of the same company product of the same volume and thus are relatively parallel experimental conditions and comparable.
Description
Technical Field
The invention belongs to the technical field of regenerative medicine, and particularly relates to an amplification culture process for large-scale preparation of billion-grade adipose-derived regenerative cells, in particular to an industrialized amplification of adipose-derived regenerative cells by combining adherent culture of a fixed packed bed with a cell batch inoculation process and a fed-batch perfusion process.
Background
The adult stem cells have the capacity of self-replication and differentiation to various cells, and can repair damaged and aged tissues of a human body, so that the adult stem cells have very important prospects in regenerative medicine clinical application, and particularly the mesenchymal stem cells and the antibody targeted drugs are rapidly developed, so that a plurality of diseases which cannot be treated originally are expected to be overcome. With the transformation of research results to clinical applications, the market demand for cells and their cellular products has increased dramatically. In order to achieve the commercial or large-scale or industrial production of cells and their additional products, the optimization and improvement of the prior art process are urgently needed to ensure the uniform quality of the prepared cells and reduce the cost to make the product price competitive in the market.
Adipose-derived mesenchymal stem cells are different from mesenchymal stem cells of other sources, such as mesenchymal stem cells of bone marrow and umbilical cord blood sources, which have been applied and accumulated for more than half a century, while adipose-derived mesenchymal stem cells begin to be started about 15 years ago, and because the adipose-derived mesenchymal stem cells are more than 1000 times more abundant than other mesenchymal stem cells, the adipose-derived mesenchymal stem cells become the most available candidate cells with the most clinical application value, and enter a development express way in 5-10 years. Due to the heterogenous characteristics of the cells, the size of the cells is several times larger than that of the mesenchymal stem cells from bone marrow and umbilical cord blood, the cell inoculation density is low, the amplification speed is relatively slow, and the total amplification amount is relatively small, so that the current process for producing the mesenchymal stem cells from hundred million-class bone marrow is not suitable for adipose-derived mesenchymal stem cells. The published literature developed for the industrial production of "hundred million" grade adipose-derived stem cells is missing.
The prototype technical scheme in the prior art is mainly derived from a large-scale culture process of mammalian cells, and the closest prior technical scheme is suspension culture of microcarrier and cross-linking technology adopted for preparing antibodies and vaccines, and an electromechanical integrated bioreactor is used. In recent 5 years, in order to respond the market demand for mesenchymal stem cell preparation, the field explores large-scale adherent culture methods of mesenchymal stem cells from various tissues, such as bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, dental pulp mesenchymal stem cells in a bioreactor for microcarrier suspension culture, and the like. Among them, the most recent technology was the production data that could be implemented for large-scale "hundred million" scale culture of adipose-derived mesenchymal stem cells published in 2015 by Sha M team of Eppendorf corporation in Siddiquee K, Sha M, Billon-cellhypoxic expansion of human mesenchyme stem cells in BioBLU 5c single-usessessessels BioProcess [ J ] J]2015, 14(2) 22-31. The parameters disclosed in the literature are: the method comprises the steps of adopting a disposable culture container with a working volume of 3.75 liters, adopting a microcarrier coated by polystyrene or collagen and with a size of 175 micrometers, adopting a process of associating cells and the microcarrier in advance in proportion and inoculating the cells and the microcarrier into the culture container at one time, adopting a culture medium containing 2-4% fetal calf serum, and respectively replacing 1/2 working volume culture media in 4 th, 8 th and 12 th days, wherein the whole culture time is 18 days. The culture results were calculated as follows: the seeded cell density was 1.75X104Cells/ml, cell density after 18 days of culture was 2.4-4.3X105Cells/ml, cellsThe density is increased by 14-24 times through 18 days of culture, and the total number of harvested cells is 16 hundred million (1.6 x 10)9) The density of the collagen-coated microcarrier is 30 g/L, the total amount of the microcarrier is 90 g, and according to the specification of a collagen microcarrier product, each gram of microcarrier provides 360cm2The cell growth area of (a) is calculated, and the cell growth surface area provided by all microcarriers is-3.24 x104cm2. The conditioned medium was recovered in total about 6 liters for 18 days of culture. The search for documents and patents 7 months later in 2015 did not see that adipose-derived mesenchymal stem cell production surpassed the above results with any technique or process beyond suspension microcarriers.
About-10 hundred million (1 x 10) cells per patient per cell are required for cell therapy9) The hundred million-class culture of adipose-derived regenerative cells is a technical bottleneck in the field, and the problems in the prior art are highlighted by high cost, small harvested cell amount and small harvested amount of conditioned medium capable of generating additional value. It is well known that the largest of the sales costs of a product is a fixed cost. The most prominent cost in the cost calculation of the prior art is the 3.75 liter disposable culture vessel and the collagen-coated microcarriers, both of which are in total at least more than 4 million RMB in the Chinese market. If a patient following a wound healing requires 10 billion (1 x 10) cells per treatment9) In terms of cells, 16 million cells can only be provided to 1 patient for 2 uses, and if each patient has 10 treatments per treatment session, the cost per treatment session per patient increases by at least 20 million RMB.
The invention does not use a disposable culture container and microcarrier, replaces a fixed packed bed adherent culture process, combines a cell 'batch inoculation' process and a 'fed-batch perfusion' process, achieves the purpose of increasing the cell harvest yield by at least more than 10 times of the closest prior art, simultaneously increases the harvest yield of a conditioned medium capable of generating an additional value, and reduces the total cost of final accounting, which is a remarkable progress of the invention. Meanwhile, the cell culture container with the volume the same as that of the cell culture container in the closest prior art, namely the cell culture container with the total volume of 5 liters and the working volume of 3.75 liters is also a series product of Eppendorf company in Germany, and therefore, the cell culture container is made under relatively parallel experimental conditions and has comparability. A. It is not obvious to a person skilled in the art that the fat-derived regenerative cells, the b. "batch seeding" process, the c. fixed packed bed of sheet carriers, and the d. "fed-batch perfusion" process combine the a.b.c. d. according to the temporal and spatial requirements and achieve substantial results. Has novelty and creativity.
Disclosure of Invention
In view of the above, the present invention relates to an amplification culture process for large-scale preparation of billion grade adipose-derived regenerative cells, and more particularly to a process combining adherent culture with fixed packed bed and cell "batch inoculation" and "fed-batch perfusion" technologies.
The specific technical scheme of the invention is as follows.
The invention provides a method for inoculating fat source regenerative cells into a fixed packed bed consisting of polyester fiber/polypropylene sheet-shaped carriers for culture.
Preferably, the adipose-derived regenerative cells are mainly composed of adipose mesenchymal stem cells.
Preferably, the fixed packed bed is arranged in an electromechanical integrated bioreactor, the bioreactor comprises a computer control center and cell culture containers, and 8 cell culture containers can be simultaneously controlled by 1 computer control center. The cell culture container with the total volume of 5 liters and the working volume of 3.75 liters can be repeatedly used, and the flaky carrier can be used for the second time.
Preferably, the fixed packed bed consists of solid sheet-like carriers, and 5 to 10g of the sheet-like carriers can be added per 100ml of the packed bed volume.
Preferably, the solid sheet-like carrier is made of 50% of polyester fiber and 50% of polypropylene which are interwoven into sheets, and then the sheets are cut into round or square small sheets with the diameter of 5-10 mm.
Preferably, the solid sheet-like support provides a surface area per gram of 1200-1400cm2 cells. Each culture container with a working volume of 3.75 liters can be provided with a fixed packed bed consisting of 50 to 150 grams of sheet-shaped carriers and can provide cultured cellsCan reach 2x105cm2。
Preferably, the seeded cells have an initial density of 3x103-6x103Cells/cm 2.
Preferably, the method for inoculating the cells is a batch inoculation process, namely, the cell inoculation is completed 3-10 times, and each time interval is 1-2 days.
Preferably, culturing a batch of cells is completed for 25-40 days, during which time the medium is changed by a "fed-batch perfusion" process, i.e. 20% of the conditioned medium is aseptically removed from the system at variable times, while the system is replenished with an equal volume of fresh medium.
Preferably, "fed-batch perfusion" is performed by using feedback control feeding, i.e. the concentration of glucose and the metabolite lactose in the system is measured daily, or the osmotic pressure in the system is measured, and the time and the amount of the additional feeding are determined by taking the measured concentration as a control index.
Compared with the prior art, the method for scale expansion culture of the billion-grade mammal adipose-derived regenerative cells provided by the invention is provided. The method abandons the disposable cell culture container in the prior art, reduces the cost by adopting the reusable culture container, further reduces the cost by adopting the solid packed bed consisting of the sheet-shaped carrier which can be used for the second time as the surface supporting body for attaching the cells, and can provide the surface area of the cell culture with the highest 2x10 in the culture container with the working volume of 3.75 liters5cm2The surface area of the microcarrier is 5-10 times that of the microcarrier which can be suspended in the same volume in the prior art. The invention changes the one-time inoculation batch culture process of the cells in the prior art into multiple batch inoculation, and is matched with the fed-batch perfusion process, thereby increasing the initial inoculation density of the cells, improving the culture efficiency, increasing the recovery amount of the conditioned medium, and increasing the added value while culturing the cells in a large scale. The significant improvements of the present invention are that more cells are harvested, more conditioned media are harvested, and the overall cost is reduced compared to the prior art.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings according to the provided drawings without creative efforts.
FIG. 1 is a photograph of a solid support used in example 1.
FIG. 2 is a photograph under a microscope at 20X of the sheet-like support used in example 1.
Fig. 3 is a schematic view of a sheet-like support in example 1.
FIG. 4 is a fixed packed bed cell culture vessel with a working volume of 3.75 liters used in examples 2, 3, 4.
Fig. 5 is a result of cell staining in example 5 in which adipose-derived mesenchymal stem cells were induced to differentiate into fat droplets.
Fig. 6 is a result of cell staining in example 5 in which adipose mesenchymal stem cells were induced to differentiate into calcified bone tissue.
FIG. 7 shows the results of cell seeding and culturing in examples 2, 3 and 4, and comparison with the closest prior art.
Detailed Description
In order to reduce the cost of the prior art to prepare fat source regenerative cells above the billion level and simultaneously harvest more conditioned medium for cell culture, the invention provides a preparation process.
The sheet-shaped carrier is white square or round, and if the sheet-shaped carrier is round, the diameter is 5-10mm, and the sheet-shaped carrier is made of polyester fiber and polypropylene.
The sheet-shaped carrier is formed by interweaving polyester fiber and polypropylene, pores and gaps for cell adsorption and adherent culture are reserved in the middle, and 1200-1400cm can be provided for each gram of sheet-shaped carrier2For cell attachment.
The sheet-shaped carrier is composed of 50% of polyester fiber and 50% of polypropylene, and cells are attached on the fiber or between the fibers.
The method provided by the invention comprises the steps of inoculating A. fat source regenerative cells onto a closed C. fixed packed bed arranged by a sheet-shaped carrier by adopting a B. "batch inoculation" process, and adopting a D. culture medium "fed batch perfusion" process. A, B, C, D are mutual preconditions, but one is not.
Further, the flaky carrier is placed into a cell culture container of a bioreactor with the working volume of 3-5 liters, the upper part and the lower part of the flaky carrier are respectively fixed by a reticular isolation plate in the container to form a fixed packed bed with the volume of 2 liters, and 3-4 liters of PBS phosphate buffer solution is injected into the container to immerse the flaky carrier.
Further, the fixed bed was immersed in PBS phosphate buffer solution, static, and fixed.
Furthermore, a magnetic stirrer is arranged in the cell culture container, and can be used for continuous or intermittent stirring.
Further, the container with the sheet-like carrier is wrapped integrally, put into an autoclave for autoclaving, cooled and connected with a computer control center according to instructions to prepare for cell inoculation.
Furthermore, the adipose-derived regenerative cells comprise a mixture of mesenchymal progenitor cells, preadipocytes, a third group of cells, adipose mesenchymal stem cells, hematopoietic cells, immune cells, epithelial cells and the like which can grow adherently, wherein the adipose mesenchymal stem cells are used as a main component, and the adipose-derived regenerative cells which have the differentiation potential and are mainly the adipose mesenchymal stem cells and have the single fiber cell form are gradually formed through adherent culture.
The channel for obtaining the cells of the present invention may be extracted from adipose tissue by a technique familiar to those skilled in the art, or may be a commercially available source.
Further, PBS phosphate buffer solution was removed according to the instructions of the apparatus, and 3.75L of culture medium, which was commercially available DMEM/F12 basal medium supplemented with 2-10% fetal bovine serum or commercially available phenol red-free and serum-free medium, was introduced.
Further, the inoculated cells are inoculated for the first time with 3x103-6x103Cells/cm 2.
Further, the introduction and removal is via an internal sterile catheter connected to a peristaltic pump.
Further, a "batch inoculation" process is employed, which means that the inoculation is repeated every 1-2 days for a total of 3-10 inoculations.
Furthermore, the fed-batch perfusion process is adopted, namely the conditioned medium with the total volume of 20% is led out, and meanwhile, the fresh medium with the same volume preheated to 37 ℃ is led in.
Furthermore, the cell culture time of each batch is 25-40 days.
Further, after the batch culture is finished, cell counting is to lead out all culture media, introduce PBS preheated to 37 ℃ in the same volume for 5 minutes, repeat twice, lead out PBS, introduce Trypsin Trypsin-EDTA preheated to 37 ℃, the volume just does not exceed a packed bed, keep at 37 ℃ for 10 minutes, add Trypsin neutralizing liquid in the same volume for 2 minutes, stir at a magnetic stirrer gap for 2-5 seconds each time, lead out all liquid, introduce PBS preheated to 37 ℃ for washing, lead out PBS, repeat 2-3 times, collect all leading-out liquid in a centralized way, divide into 250ml sterile centrifuge tubes, set up a centrifugal force of 200 and 350g on a centrifuge, centrifuge for 10 minutes, suspend the collected cells in the culture media, dilute by 10 times and 100 times, count by a blood counting plate respectively. This is a routine operation for the person skilled in the art.
Further, cells were plated at 1.8X104Cells/cm2The cells were seeded in 6-well cell culture plates for adipogenic and osteogenic differentiation experiments to verify that the sternness of the cells was not lost throughout the culture.
Further, the adipogenic differentiation test and the osteogenic differentiation test were conducted according to the instructions of the kit manufacturer ATCC, and finally photographed by an inverted microscope. This is a routine operation for the person skilled in the art.
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
The reagents used in the following examples are all commercially available reagents and kits routinely purchased by those skilled in the art: NaHCO3, DMEM/F12 basal medium, fetal bovine serum, PBS phosphate buffer solution, Trypsin Trypsin-EDTA, Trypsin neutralizing solution, fat-forming induction culture kit, osteogenesis induction culture kit and blood glucose detector, which are purchased from Sigma and ATCC company in the United states. The sheet-like carriers were purchased from Eppendorf, Inc. and Wuhansaike science and technology, Inc. in USA. Phenol red-free and serum-free human mesenchymal stem cell medium PB2004Y was purchased from tianjin scientific and technical limited for tertiary sea biologicals. The bioreactor contained a cell culture vessel with a working volume of 3.75 liters and a computer control center, both purchased from Eppendorf, USA.
Example 1.
Sheet-like support and preparation.
Weighing 50-150 g of the flaky carrier which is used for the first time or the second time repeatedly, washing with ultrapure water, and draining.
And (5) putting the mixture into a drying oven at 60 ℃ for 24 hours to completely dry.
Every 150 g of the sheet-shaped carrier is added into a cell culture container with the working volume of 3.75 liters in a German Eppendorf bioreactor, and the upper part and the lower part are respectively fixed by a mesh plate to form a fixed packed bed with the volume of about 3 liters.
Purified water is injected into the peripheral isolation interlayer of the reaction vessel, and the water amount is half of the volume of the total interlayer. To the reaction vessel, 3.75 liters of 0.01M phosphate buffer was added. All other parts are installed, fixed and packaged according to the specification of a manufacturer.
The whole fixed bed reaction vessel was transferred to an autoclave and steam sterilized at 121 ℃ for 30 minutes under high pressure.
After cooling to room temperature, the reaction vessel was connected to a computer control center, 3.75 liters of 0.01M phosphate buffer was removed via a built-in sterile catheter connected to a peristaltic pump, and 3.75 liters of medium was subsequently introduced.
The cells were ready for seeding 24 hours after the pilot run.
Example 2.
150 g of the second re-used sheet support constituted a 3 litre fixed packed bed.
Calculating the surface area provided by the sheet-like support: providing 1200cm per gram of sheet-like support 2150 grams of the sheet-like support used had a surface area of 1.8x105cm2Surface area of (a).
The cell culture medium is DMEM/F12 basic medium and 10% fetal bovine serum.
After inoculation, the rotation speed of the magnetic stirrer is set to be 25rpm, stirring is carried out for 5 minutes, the stirring is stopped for 25 minutes, and the process is repeated for 4 times, namely after 2 hours, the rotation speed of the magnetic stirrer is set to be continuous 25rpm, the stirring is maintained for 24 hours, and the pH value of the system is maintained in a range of 7.2-7.4.
The first 5 days, 3 times of total inoculation, total cell inoculation amount of 1.12x109Fixed packed bed volume 3L, final inoculum density 5.4X105Cells/ml.
From day 4, aseptically withdrawing 20ml of cell culture supernatant every day, offline detecting pH with a pH meter, introducing 5.5% NaHCO3 to adjust pH to 7.2-7.4 when pH is lower than 7.2, offline detecting glucose consumption and lactose accumulation of metabolites, timely withdrawing 50% volume of conditioned medium when glucose concentration is lower than 0.5 g/L or lactose metabolite concentration exceeds 60mM, and immediately introducing fresh medium with equal volume preheated at 37 ℃.
From day 6, the medium was changed to half volume of DMEM/F12 basal medium plus 4% fetal bovine serum and half volume of commercial phenol red-free serum-free medium.
From day 8, every 2 days 20% of the total volume of conditioned medium was removed and an equal volume of fresh medium pre-warmed at 37 ℃ was introduced.
After 25 days of culture, the whole volume of 3.75 liters of conditioned medium was removed on day 25, washed for 5 minutes with an equal volume of PBS preheated to 37 deg.C, according to the specific embodiment [0045 ]]The cells were collected and counted: end point-total number of cells 3x1010Cell density 1.5x107/ml, cell density 5.4X10 after three inoculations5The cells/ml were expanded 28-fold, with an efficiency of expansion of 1.17-2 fold compared to "14-fold expansion compared to inoculation" mentioned in the published literature in the art after 2015.
At the end of 25 days of culture, a total of more than 10.5 liters of conditioned medium was collected.
Example 3.
50g of the sheet-like support constitute a fixed packed bed of 1 liter.
Calculation of the surface area provided by the sheet-like support calculation: providing 1200cm per gram of sheet-like support250g of a sheet-like support having a surface area of 6X104cm2Surface area of (a).
The cell culture medium is DMEM/F12 basic medium and 10% fetal bovine serum.
After inoculation, the rotation speed of the magnetic stirrer is set to be 25rpm, stirring is carried out for 5 minutes, the stirring is stopped for 25 minutes, and the process is repeated for 4 times, namely after 2 hours, the rotation speed of the magnetic stirrer is set to be continuous 25rpm, the stirring is maintained for 24 hours, and the pH value of the system is maintained in a range of 7.2-7.4.
For 3 consecutive days, cell confluenceThe quantity is 1.08x109Fixed packed bed volume 1L, final inoculum density 1.08X106/ml。
On day 4, 20ml of cell culture supernatant was aseptically removed every day, the pH was measured offline using a pH meter, and when the pH was lower than 7.2, 5.5% NaHCO3 was introduced to adjust the pH to a range of 7.2 to 7.4, while the glucose consumption status and the metabolite lactose accumulation status were measured offline, and when the glucose concentration was lower than 0.5 g/l or the lactose metabolite concentration exceeded 60mM, 50% volume of conditioned medium was immediately removed, followed by introduction of an equivalent volume of fresh medium.
From day 4, the medium was changed to half the volume of DMEM/F12 basal medium plus 4% fetal bovine serum and half the volume of commercial phenol red-free serum-free medium.
From day 6, every two days, 20% of the total volume of conditioned medium was removed and an equal volume of fresh medium was introduced.
After 25 days of incubation, the whole medium was removed, washed for 5 minutes with an equal volume of PBS pre-warmed at 37 ℃ [0045 ] according to the specific embodiment]The cells were collected and counted: end-total number of cells 4.5x1010Cell density 4.5x107/ml, 1.08X10 when compared to inoculation6The amplification was 42-fold at/ml, and the efficiency of amplification was 1.75-3-fold compared to "14-24-fold amplification compared to inoculation" mentioned in the published literature in the art after 2015.
At the end of 25 days of culture, a total of greater than 11.25 liters of conditioned medium was collected.
Example 4.
50g of the sheet-like support constitute a fixed packed bed of 1 liter.
Calculating the surface area provided by the sheet-like support: providing 1200cm per gram of sheet-like support250g of a sheet-like support having a surface area of 6X104cm2Surface area of (a).
The cell culture medium is DMEM/F12 basic medium and 10% fetal bovine serum.
After inoculation, the rotation speed of the magnetic stirrer is set to be 25rpm, stirring is carried out for 5 minutes, the stirring is stopped for 25 minutes, and the process is repeated for 4 times, namely after 2 hours, the rotation speed of the magnetic stirrer is set to be continuous 25rpm, the stirring is maintained for 24 hours, and the pH value of the system is maintained in a range of 7.2-7.4.
For 10 consecutive days, the total cell inoculum size was 1.8 × 109Fixed packed bed volume 1L, inoculum density 1.8X106/ml。
On day 4, 20ml of cell culture supernatant was aseptically removed every day, and the pH was measured off-line using a pH meter, and when the pH was lower than 7.2, 5.5% NaHCO3 was introduced to adjust the pH to maintain the pH in the range of 7.2 to 7.4, while the glucose consumption status and the lactose accumulation status of the metabolite were measured off-line, and there was no case where the glucose concentration was lower than 0.5 g/l, or the lactose metabolite concentration exceeded 60 mM.
From day 11, the medium was changed to a commercial phenol red-free and serum-free medium.
From day 12, 20% of the total volume of conditioned medium was removed each day and an equal volume of fresh medium was introduced.
After 40 days of incubation, the whole medium was removed, washed for 5 minutes with an equal volume of PBS pre-warmed at 37 ℃ [0045 ] according to the specific embodiment]The cells were collected and counted: end point-cell density 4.62x107Total number of cells 4.62X 10/ml10Cell density 1.8X10 after 10 days of inoculation6The amplification efficiency is 1.08-1.86 times that of the amplification of 14 times compared with the amplification of the inoculation, which is mentioned in the published literature in the field after 2015.
At the end of 40 days of culture, a total of more than 31.5 liters of conditioned medium was collected.
Example 5.
The adipose-derived mesenchymal stem cells obtained in example 4 were collected for adipogenic induction differentiation, and the differentiation was performed according to the adipogenic differentiation kit instructions purchased from ATCC, which can be performed by those skilled in the art, and it was identified that the lipid droplets formed were stained with oil red O, and the differentiation into lipid droplets indicated that the cells after large-scale amplification remained dry, and the differentiation potential thereof was not damaged during the amplification process.
The adipose-derived mesenchymal stem cells obtained in example 4 were collected for osteogenic induction differentiation, and the differentiation was performed according to the osteogenic differentiation kit instructions purchased from ATCC, and those skilled in the art can perform the procedures to identify the bone formation condition by staining the calcified nodule with alizarin red, and differentiate to form calcified bone tissue, which indicates that the cells after large-scale amplification still remain dry, and the differentiation potential thereof is not damaged during the amplification process.
Claims (8)
1. A method for expanding adipose-derived regenerative cells to a billion level is characterized in that the adipose-derived regenerative cells are inoculated into a fixed packed bed arranged by a sheet-shaped carrier in a cell culture container by adopting a batch inoculation method, the culture medium is replaced by adopting fed-batch perfusion of the culture medium, and the total number of the cells is expanded to the billion level or more in the fixed packed bed cell culture container with the working volume of 3.75 liters; the cell batch inoculation method is that the total cell inoculation amount is divided into 3-10 times of batch inoculation, and the inoculation interval time is 1-2 days; when the cells are inoculated for the first time, the surface area for adhering and growing the cells can be provided by the sheet-shaped carrier every squareCentimeter inoculation 3x103-6x103After completion of the "batch inoculation" of individual cells, 9X10 cells were inoculated per square centimeter3-3x104And (4) cells.
2. The method of claim 1, wherein after completion of the whole inoculation, the cell culture medium is changed from a medium containing fetal bovine serum during the inoculation to a medium containing fetal bovine serum half its content or completely free of serum.
3. The method of claim 1, wherein the cells are seeded for the first time in a fixed packed bed volume of sheet support at 1.8-3.6x10 per ml volume5After completion of the "batch inoculation" of individual cells, 5.4X10 cells were inoculated per ml volume5-1 .8x106And (4) cells.
4. The method of claim 1, wherein the cell culture time is 25-40 days.
5. The method according to claim 1, wherein the "fed-batch perfusion" method comprises aseptically withdrawing conditioned medium in a volume of 20-50% of the working volume of the cell culture vessel each time, while introducing an equivalent volume of fresh medium preheated to 37 ℃ at an interval of 1-2 days.
6. The method of claim 1, wherein the cell culture vessel has a working volume of 3.75 liters and a fixed packed bed disposed therein having a volume of 1 to 3 liters.
7. The method of claim 1, wherein the sheet-like support is newly purchased for a first use or is reused for a second time.
8. The method of claim 1, wherein the adipose-derived regenerative cells comprise a mixture of adherently growing mesenchymal progenitor cells, preadipocytes, a third group of cells, adipose mesenchymal stem cells, hematopoietic cells, immune cells, cells and epithelial cells, wherein adipose mesenchymal stem cells are used as a main component, and the adipose mesenchymal stem cells with differentiation potential are gradually formed into adipose mesenchymal stem cell-based regenerative cells with single fiber cell morphology through adherent culture.
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