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CN-122012389-A - Stem cell large-scale amplification method based on microcarrier-shake flask coupling culture

CN122012389ACN 122012389 ACN122012389 ACN 122012389ACN-122012389-A

Abstract

The invention discloses a stem cell large-scale amplification method based on microcarrier-shake flask coupling culture, and belongs to the technical field of biology. The method comprises the steps of adding the pretreated microcarrier and stem cell suspension into a shake flask containing a culture medium to form an initial culture system, carrying out dynamic attachment culture to attach stem cells on the surface of the microcarrier, switching to a constant rotating speed for amplification culture after attachment is completed, and harvesting the stem cells after the amplification end point of the culture. The invention systematically solves the key technical bottlenecks of low efficiency, high cost, poor scalability and the like faced by the large-scale stem cell expansion in the prior art by constructing a set of highly integrated process flow which is simple and convenient to operate and has strong process controllability.

Inventors

  • Jia Lijunpeng
  • LI ZHONGHAN
  • Zheng Zuokang
  • WEN XIN

Assignees

  • 四川大学

Dates

Publication Date
20260512
Application Date
20260325

Claims (10)

  1. 1. The stem cell large-scale amplification method based on microcarrier-shake flask coupling culture is characterized by comprising the following steps of: S1, adding the pretreated microcarrier and the stem cell suspension into a shake flask containing a culture medium to form an initial culture system, wherein the dosage of the microcarrier is 1-5g/L, and the inoculation amount of the stem cells is 5X 10 4 ~2×10 5 cells/mL; S2, carrying out dynamic attachment culture to attach stem cells to the surface of the microcarrier, wherein the dynamic attachment culture process comprises the following steps of shaking at a speed of 80-120 rpm for 7-9 min under the conditions of 37 ℃ and 5% CO 2 , then standing for 20-25 min, defining a shaking-standing cycle as 1 period, and continuously carrying out 45-50 periods; s3, after the attachment is completed, switching to a constant rotation speed for amplification culture, and realizing large-scale amplification of stem cells; s4, after culturing to an amplification end point, harvesting stem cells.
  2. 2. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, wherein the culture medium does not induce directional differentiation of stem cells and can maintain the undifferentiated state and high proliferation activity of stem cells.
  3. 3. The method for amplifying stem cells on a large scale based on microcarrier-shake flask coupled culture according to claim 1, wherein the stem cells are SCL stem cells, and the culture medium for the SCL stem cells comprises the following components in 1 mL: 459. Mu.L of IMDM basal medium, F12 basal medium 459. Mu.L, 1. Mu.L of thioglycerol, 1. Mu.L of transferrin, Insulin 1.5. Mu.L of the solution, The amount of lipid was 10. Mu.L, Penicillin/streptomycin 10 mul, 40. Mu.L of polyvinyl alcohol, 10 Mu L of the serum replacement was knocked out, Bovine serum albumin 5 mu L, SMO receptor agonists 1 μl, 1 Mu L of the ROCK inhibitor, 1. Mu.L of the BMP inhibitor, Alkaline fibroblast growth factor 0.5. Mu.L.
  4. 4. The method for amplifying stem cells on a large scale based on microcarrier-shake flask coupled culture according to claim 1, wherein the microcarrier is a Cytodex 1 microcarrier.
  5. 5. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, wherein the microcarrier pretreatment step comprises hydration, washing, sterilization and equilibration steps performed sequentially.
  6. 6. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, wherein in step S1, the amount of microcarrier is 3g/L, and the inoculation amount of stem cells is 1×10 5 cells/mL.
  7. 7. The method for amplifying stem cells on a large scale based on microcarrier-shake flask coupled culture according to claim 1, wherein the dynamic attachment culture procedure is that the stem cells are firstly shaken at 100rpm for 8min and then left for 22min under the conditions of 37 ℃ and 5% CO 2 , and the shaking-standing cycle is defined as 1 cycle and 48 cycles are continuously performed.
  8. 8. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, wherein in step S3, continuous expansion culture is performed at a constant rotation speed of 100rpm, and half volume of fresh medium is replaced every 48 hours.
  9. 9. The method for large-scale expansion of stem cells based on microcarrier-shake flask coupled culture according to claim 1, wherein in the step S4, after expansion is finished, cell expansion culture is stopped, cell-microcarrier complex is settled, the culture medium is discarded, the cell is washed by a calcium-free magnesium buffer solution containing EDTA, a trypsin solution is added for digestion treatment, cells are dissociated from microcarriers, then a serum-containing culture medium is added for stopping digestion reaction, free cells are separated from undissociated microcarriers, and the obtained cell suspension is collected, and the cell suspension is centrifugally washed and can be used for subsequent experiments or subculture.
  10. 10. The large-scale stem cell amplification method based on microcarrier-shake flask coupling culture according to claim 9, wherein the EDTA-containing calcium-magnesium-free buffer solution is PBS with pH of 7.6, wherein the EDTA concentration is 0.02wt%, the trypsin solution is 30-50 mL/g microcarrier, and the trypsin solution is incubated for 10-20min at 37 ℃; the free cells were separated from the undissociated microcarriers by means of sedimentation by standing or sieving through a 100 μm cell sieve.

Description

Stem cell large-scale amplification method based on microcarrier-shake flask coupling culture Technical Field The invention belongs to the technical field of biology, relates to the technical field of cell culture, and in particular relates to a stem cell large-scale amplification method based on microcarrier-shake flask coupling culture. Background Stem cells have broad application prospects in the fields of regenerative medicine and cell therapy due to their unique self-renewal capacity and multidirectional differentiation potential, and have been explored for treating various refractory diseases such as blood system diseases, nervous system degenerative diseases, cardiovascular injuries, diabetes and the like. However, the key premise of transforming stem cells from laboratory studies to clinical grade therapeutic products is the stable availability of cell preparations of sufficient quantity (typically up to 10 8–1010 grade), of controlled quality, of uniform function and of compliance with safety standards. Therefore, the efficient and reliable in-vitro amplification technology forms a core link of the clinical transformation of stem cells. In the prior art, aiming at mesenchymal stem cells, hematopoietic stem cells and other adherence-dependent stem cells, the following amplification methods are mainly adopted: (1) Static two-dimensional culture methods, including conventional flask/dish subculture and multi-layered Cell Factory (Cell Factory) systems. The method relies on the adherent growth of cells on the surface of plastic, and amplification is realized through periodic pancreatin digestion and manual passage. Although the operation is simple and convenient, the method is limited by the two-dimensional surface area, the contact inhibition effect and the frequent manual intervention, and the real mass production is difficult to realize. (2) Feeder co-culture systems mimic the in vivo microenvironment to maintain stem cell viability by introducing endothelial cells or stromal cells as support layers. The method has remarkable effect on the aspect of amplifying original hematopoietic stem cells (such as CD34 +、CD38- subgroup), but has the problems of complex composition, large batch difference, potential pathogen pollution, high regulatory compliance risk and the like due to the introduction of heterologous or allogeneic cells. (3) The bioreactor combines microcarrier culture technology, utilizes suspended microcarrier to provide a three-dimensional attachment surface, and realizes uniform transmission of nutrition and gas by stirring. The system has good process controllability and amplification potential, and is suitable for automatic closed production. However, the shearing force of the fluid generated by stirring may damage cells, and the high equipment cost and complex process development limit the popularization and application of the device in small and medium-sized institutions. (4) Medium composition optimization strategies include serum-free or chemically defined medium, small molecule agonists (e.g., UM171, SR1, nicotinamide, etc.) or synthetic polymers (e.g., PVA) in place of serum proteins. Such methods help to increase the efficiency of expansion and reduce the risk of xeno, the FDA has approved the first nicotinamide-expansion-based hematopoietic stem cell product Omisirge (omidubicel-onlv). However, even so, the performance of a physical culture platform lacking a match is limited by the inherent bottleneck of conventional static culture. Although the above technology has driven the development of stem cell expansion to a varying extent, its inherent limitations severely limit the clinical transformation process, which is embodied in four aspects: First, the large-scale amplification efficiency is low. Static two-dimensional culture is limited in specific surface area and contact inhibition, and even with multi-layered cell factories, it is difficult to break through the production ceiling. For example, adipose-derived mesenchymal stem cells often enter senescence stasis after passing to the P6-P8 generation, whereas stroma-free culture systems have a much lower expansion of primitive hematopoietic stem cells than co-culture systems, failing to meet the cell dose required for adult transplantation. Second, cell product quality and functional uniformity are difficult to guarantee. Long-term expansion in vitro tends to result in phenotypic drift of stem cells (e.g., decreased expression of markers such as CD34, CD73, etc.), reduced differentiation potential, and reduced homing and regeneration capacity in vivo. More seriously, in static culture, the maldistribution of nutrients/metabolites forms a micro-environmental gradient, the inherent randomness of cells is superimposed, and the population heterogeneity is greatly amplified. This uncontrolled heterogeneity not only affects efficacy consistency. Thirdly, the process has poor controllability, high pollution risk and high cost. The tr