WO2021217297A1 - 一种制备"百亿"级脂肪源再生细胞的生产工艺 - Google Patents

一种制备"百亿"级脂肪源再生细胞的生产工艺 Download PDF

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WO2021217297A1
WO2021217297A1 PCT/CN2020/087004 CN2020087004W WO2021217297A1 WO 2021217297 A1 WO2021217297 A1 WO 2021217297A1 CN 2020087004 W CN2020087004 W CN 2020087004W WO 2021217297 A1 WO2021217297 A1 WO 2021217297A1
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cells
cell
inoculation
volume
adipose
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周莹
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深圳阿尔法生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/04—Tissue, human, animal or plant cell, or virus culture apparatus with means providing thin layers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

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  • the invention belongs to the technical field of regenerative medicine, and specifically relates to a large-scale expansion and culture process for preparing "tens of billions" fat-derived regenerative cells, and in particular to an adherent culture using a fixed packed bed combined with cell “batch inoculation” And “fed-batch perfusion” process, industrially amplifying fat-derived regenerative cells.
  • Adipose-derived mesenchymal stem cells are different from other sources of mesenchymal stem cells.
  • bone marrow and umbilical cord blood-derived mesenchymal stem cells have been used and accumulated for more than half a century, while adipose-derived mesenchymal stem cells started about 15 years ago.
  • the source is more than 1,000 times more abundant than other mesenchymal stem cells, it has become the most available candidate cell with the most clinical application value.
  • it has entered the fast lane of development. Due to its heterogeneous nature, the cell itself is several times larger than mesenchymal stem cells derived from bone marrow and cord blood.
  • the cell seeding density is low, the expansion speed is relatively slow, and the total expansion is relatively small, making the current production of "100 million”
  • the process of grade bone marrow-derived mesenchymal stem cells is not suitable for adipose-derived mesenchymal stem cells.
  • the public literature developed for the industrial production of "100 million" adipose-derived stem cells is missing.
  • the prototype technical solutions of the prior art are mainly derived from the large-scale culture process in mammalian cells.
  • the closest prior technical solutions are the suspension culture of microcarriers and cross-linking technology used in the preparation of antibodies and vaccines, and the use of mechatronics organisms. reactor.
  • this field has explored large-scale adherent culture methods of mesenchymal stem cells derived 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 are cultured in microcarrier suspension in a bioreactor, etc.
  • the process of sexual inoculation into a culture vessel is to use a medium containing 2-4% fetal bovine serum, and replace the 1/2 working volume of the medium on the 4th, 8th, and 12th day respectively.
  • the whole culture time is 18 days.
  • the cell density of inoculation is 1.75 ⁇ 10 4 cells/ml
  • the cell density after 18 days of culture is 2.4-4.3 ⁇ 10 5 cells/ml
  • the cell density has increased by 14-24 times through 18 days of culture
  • the total number of harvested cells is 1.6 billion (1.6 x10 9 )
  • the density of microcarriers coated with collagen is 30 g/liter
  • the total amount of microcarriers used is ⁇ 90 g.
  • each gram of microcarriers provides a cell growth area of ⁇ 360cm 2.
  • the surface area that the carrier can provide for cell growth is ⁇ 3.24 ⁇ 10 4 cm 2.
  • a total of about 6 liters of conditioned medium is recovered after 18 days of culture.” Searching the documents and patents after July 2015 did not find that the production of fat-derived mesenchymal stem cells uses any technology and process other than suspended microcarriers to surpass the above results.
  • each patient needs about ⁇ 1 billion (1x10 9 ) cells per time.
  • the "billion" level culture of fat-derived regenerative cells has always been a technical bottleneck in this field.
  • the problems of the existing technology are prominent in the high cost and harvest
  • the amount of cells is small, and the amount of conditioned medium that can produce added value is small.
  • fixed costs account for the largest proportion of product sales costs.
  • the most prominent cost in the cost calculation of the prior art is the 3.75-liter disposable culture container and the collagen-coated micro-carriers.
  • the total inquiries in the Chinese market are at least more than 40,000 RMB. If one patient with wound healing requires ⁇ 1 billion ( 1 ⁇ 10 9 ) cells for one cell therapy, 1.6 billion cells can only be provided to 1 patient for 2 times. If each patient uses 10 times per course, each patient The cost of each course of treatment has increased by at least RMB 200,000.
  • the present invention does not use disposable culture containers and microcarriers, instead of using a fixed packed bed adherent culture process, and combines cell "batch inoculation" and "fed-batch perfusion” processes to achieve at least the closest increase in cell harvest. It is more than 10 times that of technology, and at the same time, it increases the yield of the conditioned medium that can produce added value, and reduces the total cost of final accounting. This is a significant advancement of the present invention.
  • the present invention uses a cell culture container with the same volume as the closest prior art, that is, a cell culture container with a total volume of 5 liters and a working volume of 3.75 liters, both of which are a series of products of the German Eppendorf company, so they are in relatively parallel experimental conditions.
  • D. "Fed-batch perfusion” process which combines ABCD according to time and space requirements and obtains the essence The sexual effect is not obvious to those skilled in the art. Have novelty and creativity.
  • the present invention relates to a large-scale production process for the expansion and culture of "tens of billions of" fat-derived regenerative cells, and in particular to an adherent culture using a fixed packed bed combined with cell “batch inoculation” and “batch inoculation”. "Fed-batch perfusion" technology.
  • the invention provides a method for inoculating fat-derived regenerative cells into a fixed packed bed composed of a polyester fiber/polypropylene sheet carrier for culturing.
  • the adipose-derived regenerative cells are mainly composed of adipose-derived mesenchymal stem cells.
  • the fixed packed bed is set in a mechatronics bioreactor
  • the reactor includes a computer control center and a cell culture container, and one computer control center can control 8 cell culture containers at the same time.
  • the cell culture container with a total volume of 5 liters and a working volume of 3.75 liters selected in the present invention can be used repeatedly, and the sheet-shaped carrier can be used twice.
  • the fixed packed bed is composed of a solid flake carrier, and 5-10 g of flake carrier can be added per 100 ml of the packed bed volume.
  • the material of the solid sheet carrier is 50% polyester fiber and 50% polypropylene interwoven into a sheet shape, and then cut into a round or square small sheet with a diameter of 5-10 mm.
  • the solid sheet-like carrier can provide a surface area of 1200-1400 cm2 for cell attachment per gram.
  • Each culture culture container with a working volume of 3.75 liters can be equipped with a fixed packed bed composed of 50-150 grams of sheet carriers, which can provide a total area of cultured cells up to 2x10 5 cm 2 .
  • the initial density of the seeded cells is 3 ⁇ 10 3 -6 ⁇ 10 3 cells/cm 2.
  • the method of seeding cells is a "batch seeding" process, that is, the cell seeding is completed in 3-10 times, with an interval of 1-2 days between each time.
  • culturing a batch of cells is completed in 25-40 days, during which the medium is replaced by the "fed-batch perfusion" process, that is, 20% of the conditioned medium is aseptically exported from the system from time to time, and the same is added to the system at the same time Volume of new medium.
  • fed-batch perfusion adopts feedback-controlled feeding, that is, the concentration of glucose and metabolite lactose in the system is measured daily, or the osmotic pressure in the system is measured, and this is used as a control indicator to determine additional feeding The time and amount of replenishment.
  • the present invention provides a method for large-scale expansion and cultivation of mammalian fat-derived regenerative cells at the "tens of billions" level.
  • This method abandons the disposable cell culture container in the prior art, uses a reusable culture container to reduce the cost, and uses a solid packed bed composed of a reusable sheet carrier as a surface carrier for cell attachment, and further The cost is reduced.
  • the packed bed in a culture vessel with a working volume of 3.75 liters can provide a cell culture surface area of up to 2x10 5 cm 2 , which is 5 times the surface area provided by the suspended microcarriers in the same volume used in the prior art. ⁇ 10 times.
  • the present invention transforms the prior art cell inoculation batch culture process into multiple "batch inoculation", and cooperates with the "fed-batch perfusion" process to increase the initial cell inoculation density, increase the culture efficiency, and increase the conditions.
  • the recovery of the culture medium increases the added value while cultivating large-scale cells. Compared with the prior art, more cells are harvested, more conditioned medium is harvested, and the total cost is reduced, which is a significant progress of the present invention.
  • FIG. 1 is a physical photograph of the sheet-shaped carrier used in Example 1.
  • FIG. 1 is a physical photograph of the sheet-shaped carrier used in Example 1.
  • Figure 2 is a 20X photo of the sheet carrier used in Example 1 under a microscope.
  • FIG. 3 is a schematic diagram of the sheet carrier in Example 1.
  • FIG. 3 is a schematic diagram of the sheet carrier in Example 1.
  • Figure 4 is a fixed packed bed cell culture vessel with a working volume of 3.75 liters used in Examples 2, 3, and 4.
  • Figure 5 shows the results of cell staining in which adipose-derived mesenchymal stem cells are induced to differentiate into fat droplets in Example 5.
  • Fig. 6 shows the cell staining results of adipose-derived mesenchymal stem cells induced to differentiate into calcified bone tissue in Example 5.
  • the present invention provides a preparation process.
  • the sheet carrier is white square or round, if it is round, the diameter is 5-10mm, and the material is polyester fiber and polypropylene.
  • the sheet-shaped carrier is interwoven by polyester fiber and polypropylene, and there are pores and gaps for cells to adhere to the wall.
  • Each gram of the sheet-shaped carrier can provide a surface area of 1200-1400cm 2 for cell attachment.
  • the sheet carrier is composed of 50% polyester fiber, 50% polypropylene, and the cells are attached to or between the fibers.
  • the method provided by the present invention is to inoculate A. fat-derived regenerative cells using B. "batch inoculation” process to inoculate C. a fixed packed bed set up by a sheet carrier, and D. medium “fed-batch perfusion" Craft.
  • A.B.C.D. is a prerequisite for each other, and neither is indispensable.
  • the sheet-shaped carrier is put into a cell culture container of a bioreactor with a working volume of 3-5 liters, and the upper and lower sides are respectively fixed with a mesh isolation plate in the container to form a fixed packed bed with a volume of ⁇ 2 liters. Inject 3-4 liters of PBS phosphate buffer solution into the medium, so that the sheet-shaped carrier is immersed in it.
  • the fixed bed is immersed in PBS phosphate buffer solution, static and fixed.
  • the container containing the sheet-shaped carrier is packaged as a whole, put into an autoclave for autoclaving, and after cooling, it is connected to the computer control center according to the instructions of the instructions, and the cells are ready to be inoculated.
  • the adipose-derived regenerative cells include a mixture of mesenchymal progenitor cells, preadipocytes, a third group of cells, adipose-derived mesenchymal stem cells, hematopoietic cells, immune cells, cells, and epithelial cells that can grow adherently, wherein With adipose-derived mesenchymal stem cells as the main component, after adherent culture, adipose-derived regenerative cells with differentiation potential and adipose-derived stem cells are gradually formed in the form of single fibroblasts.
  • the cell acquisition channel of the present invention can be extracted from adipose tissue by technical means familiar to those skilled in the art, or can be a commercially available source.
  • the medium is a commercially available DMEM/F12 basal medium, plus 2-10% fetal bovine serum, or a commercially available phenol red-free Serum medium.
  • the seeded cells are seeded with 3 ⁇ 10 3 -6 ⁇ 10 3 cells/cm2 for the first time.
  • import and export operations are through the built-in sterile catheter connected to the peristaltic pump.
  • the "batch inoculation” process is adopted, which means that the inoculation is repeated once every 1-2 days, and the inoculation is 3-10 times in total.
  • adopting the "fed-batch perfusion" process refers to exporting ⁇ 20% of the total volume of the conditioned medium, and at the same time introducing the same volume of fresh medium preheated at 37°C.
  • the cell culture time of each batch is 25-40 days.
  • the cell count is to export all the culture medium, import the same volume of preheated PBS at 37°C and wash for 5 minutes, repeat twice, export the PBS, import trypsin-EDTA preheated at 37°C , The volume is just below the packed bed, keep at 37°C for 10 minutes, add the same volume of trypsin neutralization solution for 2 minutes, during which the magnetic stirrer gap stirring, every 2-5 seconds, export all the liquid, import and preheat at 37 Wash with PBS at °C, export PBS, repeat 2-3 times, after all the export solution is collected, aliquot into a 250ml sterile centrifuge tube, set a centrifugal force of 200-350g on the centrifuge, centrifuge for 10 minutes, the collected cells are suspended Dilute 10 times and 100 times in the culture medium, and count them with a hemocytometer respectively. This is a routine operation of those skilled in the art.
  • the cells were seeded into a 6-well cell culture plate at 1.8 ⁇ 10 4 cells/cm 2 to perform adipogenic differentiation and osteogenic differentiation experiments to verify that the stemness of the cells was not lost during the entire culture process.
  • the fat differentiation test and the osteogenic differentiation test were performed in accordance with the instructions of the kit manufacturer ATCC, and finally took pictures with an inverted microscope. This is a routine operation of those skilled in the art.
  • 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, pancreas Protease neutralization solution, fat induction culture kit, osteogenic induction culture kit, blood glucose detector, purchased from Sigma and ATCC in the United States.
  • the flake carrier was purchased from Eppendorf Company of the United States and Wuhan SECCO Technology Company.
  • Phenol red-free serum-free human mesenchymal stem cell medium PB2004Y was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.
  • the bioreactor including a cell culture vessel with a working volume of 3.75 liters and a computer control center were purchased from Eppendorf, USA.
  • Each 150 grams of sheet-shaped carrier is added to a cell culture vessel with a working volume of 3.75 liters in a German Eppendorf bioreactor, and the upper and lower parts are fixed with mesh plates to form a fixed packed bed with a volume of about 3 liters.
  • Calculate the surface area provided by the flake carrier provide a surface area of 1200 cm 2 per gram of the flake carrier, and the 150 g flake carrier used has a surface area of 1.8 x 10 5 cm 2.
  • the cell culture medium is DMEM/F12 basic medium and 10% fetal bovine serum.
  • the cell seeding density per unit area is 3x10 3 cells/cm 2
  • 150 g sheet carrier has a surface area of 1.8x10 5 cm 2
  • a total of 5.4x10 8 cells are seeded, and the volume is filled in 3 liters.
  • the cell seeding density per unit volume is 1.8 ⁇ 10 5 cells/ml.
  • the speed of the magnetic stirrer was set to 25 rpm, stirred for 5 minutes, stopped for 25 minutes, and repeated 4 times, that is, after 2 hours, the speed of the magnetic stirrer was set to a continuous 25 rpm for 24 hours, and the pH of the system was maintained in the range of 7.2-7.4.
  • the second cell inoculation on the third day completely repeat the inoculation on the first day.
  • the third cell inoculation on the 5th day completely repeat the inoculation on the first day.
  • the total cell inoculation amount is 1.12 ⁇ 10 9
  • the fixed packed bed volume is 3L
  • the final inoculation density is 5.4 ⁇ 10 5 cells/ml.
  • the medium was changed to half volume of DMEM/F12 basic medium plus 4% fetal bovine serum, and half volume of commercially available phenol red-free serum-free medium.
  • Calculate the surface area provided by the sheet carrier provide a surface area of 1200 cm 2 per gram of the sheet carrier, and the 50 g sheet carrier used has a surface area of 6 ⁇ 10 4 cm 2.
  • the cell culture medium is DMEM/F12 basic medium and 10% fetal bovine serum.
  • the speed of the magnetic stirrer was set to 25 rpm, stirred for 5 minutes, stopped for 25 minutes, and repeated 4 times, that is, after 2 hours, the speed of the magnetic stirrer was set to a continuous 25 rpm for 24 hours, and the pH of the system was maintained in the range of 7.2-7.4.
  • the second cell inoculation on the second day completely repeat the inoculation on the first day.
  • the third cell inoculation on day 3 completely repeat the inoculation on day 1.
  • the total cell inoculation amount was 1.08x10 9
  • the fixed packed bed volume was 1L
  • the final inoculation density was 1.08x10 6 /ml.
  • the medium was changed to half the volume of DMEM/F12 basic medium plus 4% fetal bovine serum, and half of the commercially available phenol red serum-free medium.
  • Calculate the surface area provided by the sheet carrier provide a surface area of 1200 cm 2 per gram of the sheet carrier, and the 50 g sheet carrier used has a surface area of 6 ⁇ 10 4 cm 2.
  • the cell seeding density per unit area is 3x10 3 cells/cm 2
  • 50 grams of sheet carrier has a surface area of 6x10 4 cm 2 and 1.8x10 8 cells are seeded, calculated as a packed bed with a volume of 1 liter
  • the cell seeding density per unit volume is 1.8x10 5 cells/ml.
  • the speed of the magnetic stirrer was set to 25 rpm, stirred for 5 minutes, stopped for 25 minutes, and repeated 4 times, that is, after 2 hours, the speed of the magnetic stirrer was set to a continuous 25 rpm for 24 hours, and the pH of the system was maintained in the range of 7.2-7.4.
  • the second cell inoculation on the second day completely repeat the inoculation on the first day.
  • the third cell inoculation on day 3 completely repeat the inoculation on day 1.
  • the fourth cell inoculation on the 4th day completely repeat the inoculation on the first day.
  • the fifth cell inoculation on the 5th day completely repeat the inoculation on the first day.
  • the sixth cell inoculation on the 6th day completely repeat the inoculation on the first day.
  • the seventh cell inoculation on the 7th day completely repeat the inoculation on the first day.
  • the 8th cell inoculation on the 8th day Completely repeat the inoculation on the 1st day.
  • the 9th cell inoculation on the 9th day completely repeat the inoculation on the 1st day.
  • the 10th cell inoculation on the 10th day Completely repeat the inoculation on the first day.
  • the total cell inoculation amount was 1.8x10 9
  • the fixed packed bed volume was 1L
  • the inoculation density was 1.8x10 6 /ml.
  • the medium was changed to a commercially available phenol red-free serum-free medium.
  • the adipose-derived mesenchymal stem cells obtained in Example 4 were collected for adipogenic induction and differentiation according to the instructions of the adipogenic differentiation kit purchased from ATCC. Those skilled in the art can operate and identify the formation of lipid droplets by staining with Oil Red O. Differentiation to form lipid droplets indicates that the cells after large-scale expansion are still dry, and their differentiation potential is not impaired during the expansion process.
  • the adipose-derived mesenchymal stem cells obtained in Example 4 were collected for osteogenic induction and differentiation according to the instructions of the osteogenic differentiation kit purchased from ATCC. Those skilled in the art can operate to identify the bone formation and stain calcified knots with Alizarin Red The ability to differentiate into calcified bone tissue indicates that the cells after large-scale expansion still maintain their stemness, and their differentiation potential is not impaired during the expansion process.

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Abstract

提供了一种"百亿"级脂肪源再生细胞的扩增培养方法。通过细胞的"分批接种"工艺，将脂肪源再生细胞接种并贴附到细胞培养容器中由片状载体设置的固定填充床，再结合"补料分批灌流"工艺，将现有技术的"十亿"级提高到到"百亿"级细胞扩增。

Description

一种制备“百亿”级脂肪源再生细胞的生产工艺 技术领域
本发明属于再生医学技术领域，具体涉及一种大规模制备“百亿”级脂肪源再生细胞的扩增培养工艺，特别是涉及一种运用固定填充床的贴壁培养结合细胞“分批接种”和“补料分批灌流”工艺，工业化扩增脂肪源再生细胞。
背景技术
成人干细胞具有自我复制和向多种细胞分化的能力，能修复人体损伤和衰老的组织，因而在再生医学临床应用上有非常重要的前景，尤其是间充质干细胞和抗体靶向药物的快速发展，使得很多原本无法治疗的病症有望得到攻克。随着研究成果向临床应用的转化，市场对细胞及其细胞产品的需求急剧增大。要达到商业化或规模化或工业化地生产细胞及其附加产品，要保障制备的细胞统一质量，要降低成本使产品价格在市场上有竞争力，就迫切需要对现有技术工艺进行优化和改良。
脂肪源间充质干细胞有别于其他来源的间充质干细胞，如骨髓和脐带血来源的间充质干细胞已经有半个世纪以上的应用积累，而脂肪源间充质干细胞约15年前开始起步，由于来源比其他间充质干细胞丰富1000倍以上成为最可获得的最有临床应用价值的候选细胞，近5-10年进入发展快车道。由于本身的异源特性，细胞本身大小比骨髓和脐带血来源的间充质干细胞大很几倍，细胞接种密度低，扩增速度相对慢，扩增总量相对小，使得目前生产“亿”级骨髓来源的间充质干细胞的工艺不适合脂肪源间充质干细胞。为工业化生产“亿”级脂肪源干细胞而研发的公开文献缺失。
现有技术的原型技术方案主要来源于在哺乳动物细胞的大规模培养工艺，最接近的现有技术方案有抗体及疫苗制备采用的微载体及交联技术的悬浮培养，使用机电一体化的生物反应器。近5年为响应市场对间充质干细胞制备的需求，本领域探索了各种组织来源的间充质干细胞的大规模贴壁培养方法，比如骨髓间充质干细胞，脂肪源间充质干细胞，脐带血间充质干细胞，牙髓间充质干细胞在生物反应器中进行微载体悬浮培养等。其中对脂肪源间充质干细胞的大规模“亿”级培养的可实施生产数据以Eppendorf公司的Sha M团队在2015年公开发表的文献为最接近的技术，文献是Siddiquee K,Sha M.Billion-cell hypoxic expansion of human mesenchymal stem cells in
Figure PCTCN2020087004-appb-000001
5c single-use vessels.BioProcess[J],2015；14(2):22–31.。文献中公开的参数是：“采用一次性使用的3.75升工作体积的培养容器，采用聚苯乙烯或胶原蛋白包被的～175微米大小的微载体，采用细胞和微载体事先按比例关联好一次性接种到培养容器中的工艺，采用含有2-4％胎牛血清的培养基，采用第4，8，12天分别更换1/2工作体积培养基，整个培养时间是18天。培养结果计算下来：接种细胞密度是1.75x10 4细胞/ml，培养18天后的细胞密度是2.4-4.3x10 5细胞/ml，细胞密度通过18天的培养增加了14-24倍，收获细胞总数16亿(1.6x10 9)，使用胶原蛋白包被的微载体密度30克/升，使用微载体总量～90克，根据胶原微载体产品说明书，每克微载体提供～360cm 2的细胞生长面积计算，全部微载体能提供的细胞生长的表面积为～3.24x10 4cm 2。18天培养总共回收条件培养基6升左右”。检索2015年7月以后的文献和 专利没有看到脂肪源间充质干细胞生产用优于悬浮微载体以外的任何技术和工艺来超越上述结果的。
细胞治疗中每个患者每次细胞需要量大约～10亿(1x10 9)，脂肪源再生细胞的“亿”级培养一直是本领域的技术瓶颈，现有技术的问题突出表现在成本高，收获细胞量少，能够产生附加值的条件培养基收获量少。众所周知，产品销售成本中占比最大的是固定成本费用。现有技术成本计算中最突出的成本是3.75升的一次性培养容器和胶原蛋白包被的微载体，中国市场询价二者总计至少大于4万人民币。如果按照一个创口愈合的患者一次细胞治疗需要～10亿(1x10 9)细胞来计算，16亿细胞只能提供给1个患者使用2次，如果每个患者每个疗程10次的话，每个患者每个疗程成本就这一项就增加至少20万人民币以上。
本发明不使用一次性培养容器和微载体，代替以固定填充床贴壁培养工艺，结合细胞“分批接种”和“补料分批灌流”工艺，达到增加细胞收获量至少是最接近的现有技术的10倍以上，同时增加了能够产生附加值的条件培养基的收获量，降低最终核算的总成本，这是本发明所具有的显著的进步。同时本发明采用和最接近现有技术的同样体积的细胞培养容器，即总体积5升，工作体积3.75升的细胞培养容器，同为德国Eppendorf公司的系列产品，因而是在相对平行的实验条件下做出的，具有可比性。A.脂肪源再生细胞，B.“分批接种”工艺，C.片状载体设置的固定填充床，D.“补料分批灌流”工艺，将A.B.C.D.按时间和空间要求组合起来并取得实质性效果，对本领域技术人员来说不是显而易见的。具备新颖性和创造性。
发明内容
有鉴于此，本发明涉及一种大规模制备“百亿”级脂肪源再生细胞的扩增培养的工艺，特别是涉及一种运用固定填充床的贴壁培养结合细胞“分批接种”和“补料分批灌流”技术的工艺。
本发明的具体技术方案如下。
本发明提供了一种将脂肪源再生细胞接种到由聚酯纤维/聚丙烯片状载体组成的固定填充床中进行培养的方法。
优选的，所述脂肪源再生细胞以脂肪间充质干细胞为主要成分。
优选的，所述固定填充床是设置在机电一体化的生物反应器中，反应器包括电脑控制中心和细胞培养容器，1个电脑控制中心可以同时控制8个细胞培养容器。本发明选用的总体积5升工作体积3.75升的细胞培养容器可以反复使用，片状载体可以二次使用。
优选的，所述固定填充床由固体片状载体组成，每100ml填充床体积中可加入5-10g片状载体。
优选的，所述固体片状载体的材质是50％聚酯纤维和50％聚丙烯交织成片状，再切割成直径5-10mm的圆形或方形小片状。
优选的，所述固体片状载体每克能提供1200-1400cm2细胞贴附的表面积。每个3.75升工作体积的培养培养容器能设置50-150克片状载体组成的固定填充床，能提供培养细胞的总面积最高可达到2x10 5cm 2。
优选的，所述接种的细胞的起始密度为3x10 3-6x10 3细胞/cm2。
优选的，所述接种细胞的方法为“分批接种”工艺，即细胞接种分3-10次完成，每次 间隔时间1-2天。
优选的，培养一批次细胞25-40天完成，期间更换培养基采取“补料分批灌流”工艺，即不定时从系统中无菌导出20％的条件培养基，同时向系统中补充同等体积的新培养基。
优选的，“补料分批灌流”采用有反馈控制流加，即每日测定系统中葡萄糖和代谢产物乳糖的浓度，或测定系统中的渗透压，并以此为控制指标来决定额外补料的时间和补料量。
与现有技术相比，本发明所提供的一种规模化扩增培养“百亿”级哺乳动物脂肪源再生细胞的方法。本方法放弃了现有技术中的一次性细胞培养容器，采用可重复使用的培养容器降低了成本，采用可二次使用的片状载体组成的固体填充床作为细胞贴附的表面承载体，进一步降低了成本，在工作体积3.75升的培养容器中的填充床能提供细胞培养的表面积最高达到2x10 5cm 2，是现有技术使用的同等体积里能悬浮的微载体所能提供的表面积的5～10倍。本发明将现有技术的细胞一次接种批式培养工艺改造为多次“分批接种”，配合“补料分批灌流”工艺，增加了细胞的起始接种密度，提高培养效率，增加了条件培养基的回收量，在大规模细胞培养的同时增加了附加价值。和现有技术相比收获了更多的细胞，收获了更多的条件培养基，降低了总成本，这是本发明所具有的显著进步。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单得介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为在实施例1中采用的片状载体实体照片。
图2为在实施例1中采用的片状载体在显微镜下20X的照片。
图3为在实施例1中的片状载体示意图。
图4为在实施例2，3，4中使用的工作体积为3.75升的固定填充床细胞培养容器。
图5为在实施例5中脂肪间充质干细胞诱导分化成脂肪滴的细胞染色结果。
图6为在实施例5中脂肪间充质干细胞诱导分化成钙化骨组织的细胞染色结果。
图7为在实施例2，3，4的细胞接种和培养结果，以及和最接近的现有技术的比较。
具体实施方式
为了降低现有技术成本来制备“百亿”级以上的脂肪源再生细胞，并同时收获更多的细胞培养的条件培养基，本发明提供了一种制备工艺。
片状载体为白色方形或圆形,如果是圆形，直径5-10mm,材质是聚酯纤维和聚丙烯。
片状载体由聚酯纤维和聚丙烯交织而成，中间留有供细胞吸附贴壁培养的孔隙和空隙，每克片状载体可提供1200-1400cm 2的表面积供细胞贴附。
片状载体组成是50％聚酯纤维，50％聚丙烯，细胞贴附在纤维之上或者纤维之间。
本发明提供的方法是将A.脂肪源再生细胞采用B.“分批接种”工艺接种到封闭的 C.由片状载体设置的固定填充床上，采用D.培养基“补料分批灌流”工艺。A.B.C.D.互为前提条件，缺一不可。
进一步的，将片状载体放入工作体积为3-5升的生物反应器的细胞培养容器，上下分别用容器中网状的隔离板固定住，形成～2升体积的固定填充床，往容器中注入3-4升PBS磷酸盐缓冲溶液，使片状载体浸没其中。
进一步的，所述固定床浸没在PBS磷酸盐缓冲溶液里面，静态的，固定的。
进一步的，细胞培养容器中有内置的磁力搅拌器，可以连续或间隙搅拌。
更进一步的，装有片状载体的容器，整体包裹好，放入高压灭菌锅进行高压灭菌，冷却后按说明书指示和电脑控制中心连接好，准备接种细胞。
进一步的，所述脂肪源再生细胞包括能贴壁生长的间质祖细胞，前脂肪细胞，第三组细胞，脂肪间充质干细胞，造血细胞，免疫细胞，细胞和上皮细胞等的混合物，其中以脂肪间充质干细胞为主要成分，经过贴壁培养，逐步形成单一纤维细胞形态的有分化潜能的以脂肪间充质干细胞为主的脂肪源再生细胞。
本发明对细胞的获取渠道可以是本领域技术人员熟悉的技术手段从脂肪组织中提取，也可以是市售来源。
进一步的，按仪器说明书指示导出PBS磷酸盐缓冲溶液，导入3.75升培养基，培养基为市售的DMEM/F12基础培养基，加2-10％胎牛血清，或者市售的无酚红无血清培养基。
更进一步的，所述接种细胞首次接种3x10 3-6x10 3细胞/cm2。
更进一步的，导入和导出操作是通过和蠕动泵连接的内置无菌导管。
进一步的，采用“分批接种”工艺，指每1-2天重复接种一次，总共接种3-10次。
进一步的，采用“补料分批灌流”工艺是指将～20％总体积的条件培养基导出，同时将预热在37℃的同等体积的新鲜培养基导入。
进一步的，每个批次细胞培养时间25-40天。
进一步的，批次培养结束后细胞计数是导出所有培养基，导入同等体积的预热在37℃的PBS清洗5分钟，重复两遍后导出PBS，导入预热在37℃的胰蛋白酶Trypsin-EDTA，体积刚好没过填充床，37℃保持10分钟，加入同等体积胰蛋白酶中和液，2分钟，其间磁力搅拌器间隙搅拌，每次2-5秒钟，导出所有液体，导入预热在37℃的PBS清洗，导出PBS，重复2-3次，所有导出液集中收集后，分装在250ml的无菌离心管中，在离心机上设置离心力200-350g，离心10分钟，收集到的细胞悬浮在培养基中，以10倍和100倍稀释，分别用血球计数板计数。这是本领域技术人员的常规操作。
更进一步的，将细胞以1.8x10 4细胞/cm 2接种到6孔细胞培养板中进行成脂肪分化和成骨分化实验以验证细胞的干性在整个培养过程中没有丢失。
更进一步的，成脂肪分化试验和成骨分化试验按照试剂盒出厂商ATCC的说明书指示操作，最后用倒置显微镜拍照。这是本领域技术人员的常规操作。
下面将结合本发明具体实施例对本发明的技术方案进行清楚和完整的描述，显然，所描述的实施例只是本发明一部分实施例，而不是全部的实施例。本领域技术人员应当理解，对本发明的具体实施例进行修改或者对部分技术特征进行同等替换，而不脱离本发明技术方案的精神，均应该涵盖在本发明保护的范围中。
以下实施例中所使用的试剂都为本领域技术人员常规购置的市售试剂和试剂盒： NaHCO3，DMEM/F12基础培养基、胎牛血清、PBS磷酸盐缓冲溶液、胰蛋白酶Trypsin-EDTA、胰蛋白酶中和液、成脂肪诱导培养试剂盒、成骨诱导培养试剂盒，血糖检测器，购自美国Sigma和ATCC公司。片状载体购自美国Eppendorf公司和武汉赛科成科技公司。无酚红无血清人间充质干细胞培养基PB2004Y购自天津灏洋生物制品科技有限公司。生物反应器包含工作体积为3.75升的细胞培养容器和电脑控制中心均购自美国Eppendorf公司。
实施例1。
片状载体和制备。
称取50-150克第一次或第二次重复使用的片状载体，超纯水洗净，控干。
放入烘干烤箱60℃，24小时，完全烘干。
按每150克片状载体加入到德国Eppendorf生物反应器中工作体积为3.75升的细胞培养容器中，上下分别用网孔板固定好，形成约3升体积的固定填充床。
将反应容器的外围隔离夹层中注入纯净水，水量为总夹层体积的一半。反应容器中加入0.01M的磷酸缓冲液3.75升。所有其他部位按厂商说明书要求安装、固定、包封好。
将上述固定床反应容器整体移入高压灭菌锅，121℃，30分钟高压蒸汽灭菌。
冷却至室温后，将反应容器和电脑控制中心连接好，通过内置的和蠕动泵相连的无菌导管将上述3.75升0.01M的磷酸缓冲液导出，随后将3.75升培养基导入。
试运行24小时后准备接种细胞。
实施例2。
150克第二次重复使用的片状载体组成3升固定填充床。
计算片状载体所提供的表面积：按每克片状载体提供1200cm 2的表面积，所用150克片状载体有1.8x10 5cm 2的表面积。
细胞培养基为DMEM/F12基础培养基和10％胎牛血清。
第1天第1次细胞接种量计算：单位面积细胞接种密度3x10 3细胞/cm 2，150克片状载体有1.8x10 5cm 2的表面积，共接种5.4x10 8细胞，按3升体积的填充床计算，单位体积细胞接种密度是1.8x10 5细胞/ml。
接种后磁力搅拌器转速设置25rpm，搅拌5分钟，停25分钟，重复4遍，即2小时后，磁力搅拌器转速设置成连续的25rpm，维持24小时，体系pH维持在7.2-7.4范围。
第3天第2次细胞接种：完全重复第1天接种情况。
第5天第3次细胞接种：完全重复第1天接种情况。
前5天，共接种3次，细胞总接种量1.12x10 9，固定填充床体积3L，最终接种密度是5.4x10 5细胞/ml。
第4天起，每天无菌导出细胞培养上清液20ml，用pH计离线检测pH值，当pH值低于7.2时，导入5.5％NaHCO3调节pH维持在7.2-7.4范围，同时离线检测葡萄糖消耗状况和代谢产物乳糖积聚状况，当葡萄糖浓度低于0.5克/升，或乳糖代谢产物浓度超过60mM时及时导出50％体积的条件培养基，随即导入同等体积预热在37℃的新鲜培养基。
从第6天开始，培养基换成一半体积的DMEM/F12基础培养基加4％胎牛血清，一半体积的市售无酚红无血清培养基。
从第8天开始，每2天导出20％总体积的条件培养基，导入同等体积预热在37℃的新鲜培养基。
培养25天结束，第25天导出全部3.75升条件培养基，导入同等体积的预热在37℃的PBS清洗5分钟，按具体实施方式所述，收集细胞并计数：终点-细胞总数3x10 10，细胞密度1.5x10 7/ml，比三次接种后细胞密度5.4x10 5细胞/ml扩增了28倍，扩增效率是2015年后本领域公开发表文献所提到的“比接种时扩增了14倍”的1.17-2倍。
培养25天结束，总共收集到条件培养基大于10.5升。
实施例3。
50克片状载体组成1升固定填充床。
计算片状载体所提供的表面积计算：按每克片状载体提供1200cm 2的表面积，所用50克片状载体有6x10 4cm 2的表面积。
细胞培养基为DMEM/F12基础培养基和10％胎牛血清。
第1天第1次细胞接种量计算：单位面积细胞接种密度6x10 3细胞/cm 2，50克片状载体有6x10 4cm 2的表面积，接种3.6x10 8细胞，按1升体积的填充床计算，单位体积细胞接种密度是3.6x10 5细胞/ml。
接种后磁力搅拌器转速设置25rpm，搅拌5分钟，停25分钟，重复4遍，即2小时后，磁力搅拌器转速设置成连续的25rpm，维持24小时，体系pH维持在7.2-7.4范围。
第2天第2次细胞接种：完全重复第1天接种情况。
第3天第3次细胞接种：完全重复第1天接种情况。
连续3天，细胞总接种量1.08x10 9，固定填充床体积1L，最终接种密度是1.08x10 6/ml。
第4天，每天无菌导出细胞培养上清液20ml，用pH计离线检测pH值，当pH值低于7.2时，导入5.5％NaHCO3调节pH维持在7.2-7.4范围，同时离线检测葡萄糖消耗状况和代谢产物乳糖积聚状况，当葡萄糖浓度低于0.5克/升，或乳糖代谢产物浓度超过60mM时及时导出50％体积的条件培养基，随即导入同等体积新鲜培养基。
从第4天开始，培养基换成一半体积的DMEM/F12基础培养基加4％胎牛血清，一半体积的市售无酚红无血清培养基。
从第6天开始，每两天导出20％总体积的条件培养基，导入同等体积的新鲜培养基。
培养25天结束，导出全部培养基，导入同等体积的预热在37℃的PBS清洗5分钟，按具体实施方式所述，收集细胞并计数：终点-细胞总数4.5x10 10，细胞密度4.5x10 7/ml，比接种时1.08x10 6/ml扩增了42倍，扩增效率是2015年后本领域公开发表文献所提到的“比接种时扩增了14-24倍”的1.75-3倍。
培养25天结束，总共收集到条件培养基大于11.25升。
实施例4。
50克片状载体组成1升固定填充床。
计算片状载体所提供的表面积：按每克片状载体提供1200cm 2的表面积，所用50克片状载体有6x10 4cm 2的表面积。
细胞培养基为DMEM/F12基础培养基和10％胎牛血清。
第1天第1次细胞接种量计算：单位面积细胞接种密度3x10 3细胞/cm 2，50克片状载体有6x10 4cm 2的表面积，接种1.8x10 8细胞，按1升体积的填充床计算，单位体积细胞接种密度是1.8x10 5细胞/ml。
接种后磁力搅拌器转速设置25rpm，搅拌5分钟，停25分钟，重复4遍，即2小时后，磁力搅拌器转速设置成连续的25rpm，维持24小时，体系pH维持在7.2-7.4范围。
第2天第2次细胞接种：完全重复第1天接种情况。
第3天第3次细胞接种：完全重复第1天接种情况。
第4天第4次细胞接种：完全重复第1天接种情况。
第5天第5次细胞接种：完全重复第1天接种情况。
第6天第6次细胞接种：完全重复第1天接种情况。
第7天第7次细胞接种：完全重复第1天接种情况。
第8天第8次细胞接种：完全重复第1天接种情况。
第9天第9次细胞接种：完全重复第1天接种情况。
第10天第10次细胞接种：完全重复第1天接种情况。
连续10天，细胞总接种量1.8x10 9，固定填充床体积1L，接种密度是1.8x10 6/ml。
第4天，每天无菌导出细胞培养上清液20ml，用pH计离线检测pH值，当pH值低于7.2时，导入5.5％NaHCO3调节pH维持在7.2-7.4范围，同时离线检测葡萄糖消耗状况和代谢产物乳糖积聚状况，没有出现葡萄糖浓度低于0.5克/升，或乳糖代谢产物浓度超过60mM的情况。
从第11天开始，培养基换成市售的无酚红无血清培养基。
从第12天开始，每天导出20％总体积的条件培养基，导入同等体积的新鲜培养基。
培养40天结束，导出全部培养基，导入同等体积的预热在37℃的PBS清洗5分钟，按具体实施方式所述，收集细胞并计数：终点-细胞密度4.62x10 7/ml，细胞总数4.62x10 10，比接种10天后的细胞密度1.8x10 6/ml扩增了26倍，扩增效率是2015年后本领域公开发表文献所提到的“比接种时扩增了14倍”的1.08-1.86倍。
培养40天结束，总共收集到条件培养基大于31.5升。
实施例5。
收集实施例4得到的脂肪间充质干细胞进行成脂肪诱导分化，按照购自ATCC的成脂分化试剂盒说明书进行，本领域技术人员都可以操作，鉴定脂滴形成情况用油红O染色，能分化形成脂滴说明大规模扩增后的细胞仍然保持干性，其分化潜能没有在扩增过程中受损。
收集实施例4得到的脂肪间充质干细胞进行成骨诱导分化，按照购自ATCC的成骨分化试剂盒说明书进行，本领域技术人员都可以操作，鉴定骨形成情况用茜素红染色钙化结结，能分化形成钙化骨组织说明大规模扩增后的细胞仍然保持干性，其分化潜能没有在扩增过程中受损。

Claims (10)

  1. 一种扩增脂肪源再生细胞到“百亿”级的工艺方法，其特征在于，采用“分批接种”工艺将脂肪源再生细胞接种到细胞培养容器中由片状载体设置的固定填充床，采用培养基的“补料分批灌流”工艺更换培养基，在工作体积3.75升的固定填充床细胞培养容器中将细胞总数扩增到“百亿”级以上。
  2. 根据权利要求1所述的方法，其特征在于，所述细胞“分批接种”工艺为将细胞接种总量分为3-10次分批接种，接种间隔时间为1-2天。
  3. 根据权利要求1所述的方法，其特征在于，完成全部接种后，细胞培养基由接种期间的含胎牛血清培养基换成胎牛血清含量是原来的一半或完全无血清培养基。
  4. 根据权利要求1所述的方法，其特征在于，所述细胞首次接种时，按片状载体所能提供细胞贴附生长的表面积，每平方厘米接种3x10 3-6x10 3个细胞，“分批接种”完成后，每平方厘米接种9x10 3-3x10 4个细胞。
  5. 根据权利要求1所述的方法，其特征在于，所述细胞首次接种时，按片状载体组成的固定填充床体积，每毫升体积接种细胞1.8-3.6x10 5个细胞，“分批接种”完成后，每毫升体积接种5.4x10 5-1.8x10 6个细胞。
  6. 根据权利要求1所述的方法，其特征在于，所述细胞培养时间25-40天。
  7. 根据权利要求1所述的方法，其特征在于，“补料分批灌流”工艺是每次无菌导出条件培养基的体积为细胞培养容器工作体积的20-50％，同时导入同等体积预热37℃的新鲜培养基，间隔时间1-2天。
  8. 根据权利要求1所述的方法，其特征在于，所述细胞培养容器工作体积为3.75升，里面设置的固定填充床体积1-3升。
  9. 根据权利要求1所述的方法，其特征在于，所述片状载体是新购置第一次使用的，或者是第二次重复使用的。
  10. 根据权利要求1所述的方法，其特征在于，所述脂肪源再生细胞包括能贴壁生长的间质祖细胞，前脂肪细胞，第三组细胞，脂肪间充质干细胞，造血细胞，免疫细胞，细胞和上皮细胞等的混合物，其中以脂肪间充质干细胞为主要成分，经过贴壁培养，逐步形成单一纤维细胞形态的有分化潜能的以脂肪间充质干细胞为主的脂肪源再生细胞。
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