Culture solution for in vitro efficient amplification of animal cells and application of culture solution

An animal cell and culture medium technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of poor preservation and application of medium, high price, and high price of low-serum or serum-free medium. , to achieve the effect of clear and clear composition, avoiding batch-to-batch variation, and good proliferation potential

Inactive Publication Date: 2013-05-08
UNION STEMCELL & GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high-quality water must be used to prepare serum-free medium. Hormones and growth factors are usually added to serum-free medium, which is chemically limited and expensive. Cells are susceptible to certain mechanical factors and growth factors in serum-free medium. Influenced by chemical factors, the storage and application of the medium is not as convenient as the traditional synthetic medium, and it is highly targeted. A serum-free medium is only suitable for the cultivation of a certain type of cells
[0011] (6) At present, the widely used culture system of adherent cells mostly uses fetal bovine serum, which contains complement components, and the remaining fetal bovine serum may cause adverse reactions in patients
[0013] 1. The existing serum-free medium uses blood products whose components have not been fully determined to replace serum. Because some components are unknown, the risk of cell contamination cannot be determined, which leads to unpredictable clinical risks.
[0014] 2. The existing culture medium used to amplify the growth of adherent cells, especially stem cells, stem cells begin to differentiate after 20 generations of culture and lose the plasticity and clinical application efficiency of stem cells
[0015] 3. The existing serum-free medium formula is very complicated, and the cost of materials required is several times higher than that of animal serum, so that it cannot be widely used
[0016] 4. Commercial low-serum or serum-free media are generally expensive and not suitable for large-scale expansion of cells

Method used

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  • Culture solution for in vitro efficient amplification of animal cells and application of culture solution
  • Culture solution for in vitro efficient amplification of animal cells and application of culture solution
  • Culture solution for in vitro efficient amplification of animal cells and application of culture solution

Examples

Experimental program
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Effect test

Embodiment 1

[0046] After the umbilical cord or placenta is removed from the operating table, take it out in the ultra-clean bench, rinse the residual blood on the surface with D-PBS, add collagenase and digest it in a constant temperature oscillator, filter the cells through a 100-mesh sieve, and use D-Hank's solution Wash the cells 3 times, and put the cell suspension into T-75 cell culture flasks for culture. The components of the culture solution per liter in this example are: DMEM / F12 medium (commercially available such as GIBCO, Hyclone, Youkang Jiye Biotechnology (Beijing) Co., Ltd., etc.) added with 0.11g sodium pyruvate and 1.5g sodium bicarbonate Yes) 0.98L, 0.02L fetal bovine serum, 9ug of bFGF and 9ug of EGF, 2mmol of glutamine. Using the above culture medium, replace the culture medium every 3 days, subculture when the cells grow to 80% full, and draw the cell growth curve and cell doubling time.

[0047] Cell doubling time refers to the time required for a proliferative cell...

Embodiment 2

[0073] Under sterile conditions, the foreskin after circumcision was taken from children, and washed three times with sterile PBS; the subcutaneous tissue was cut off, and the skin piece was digested in trypsin at 37°C for 2 hours; the epidermis and dermis were mechanically separated with ophthalmic forceps; Wash the dermis three times with sterile PBS and cut it into about 1mm with ophthalmic scissors 3 Large and small tissue pieces; transfer the tissue pieces to a centrifuge tube, add collagenase solution and pipette evenly, at 37°C, 5% CO 2 Digest in the incubator for 4 hours, filter the digestion solution through a 150-mesh stainless steel filter; collect the digestion solution, centrifuge at 1000rpm for 5 minutes, discard the supernatant, and centrifuge once again in the same way; put the cell suspension into a T-75 cell culture bottle for cultivation . The components of the culture solution per liter in this example are: DMEM / F12 medium (commercially available such as G...

Embodiment 3

[0081] Recovery and culture of tumor cell lines (including HeLa cell line and HepG2 cell line): Take out the cryovial from liquid nitrogen, place it in warm water immediately and keep stirring. Thaw the frozen material in the cryovial within 1 minute. Open the cryovial and pipette the cell suspension into a centrifuge tube. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Add 10ml of culture medium to the precipitate, pipette evenly, then centrifuge for 10 minutes, and discard the supernatant. After adding the appropriate medium, transfer the cells to a culture flask at 37°C, 5% CO 2 Cultivate in an incubator and observe the growth the next day. The components of the culture solution per liter in this example are: DMEM / F12 medium with 0.11 g of sodium pyruvate and 2 g of sodium bicarbonate added (commercially available such as GIBCO, Hyclone, Youkang Jiye Biotechnology (Beijing) Co., Ltd., etc. ) 0.98L, 0.02L fetal bovine serum, 15ug EGF, 2mmol glutamine....

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Abstract

The invention discloses a culture solution for an in vitro efficient amplification of animal cells and an application of the culture solution. The culture solution consists of basal culture mediums, namely DMEM/F12, bFGF, EGF, sodium pyruvate, glutamine, sodium hydrogen carbonate and fetal calf serum. The culture solution disclosed by the invention has comparatively low content of animal serum, and is comparatively high in safety, simple and definite in formula components; the culture solution can avoid a possibility of different properties of the cultured cells due to a difference between culture medium batches, and is applicable to industrial production of cell products. Animal adherent cells cultured by the culture solution disclosed by the invention are comparatively strong in in-vitro amplification capacity and well maintain biological characteristics; and the culture solution is not only applicable to culture of adherent mesenchymal stem cells, but also suitable for culture of other fibroblast lines and other cell lines.

Description

technical field [0001] The invention belongs to the formula of a culture solution in the field of cell and tissue culture, and relates to a culture solution with low serum content and clear components, which is especially suitable for expanding adherent cells in vitro. technical background [0002] Cell culture medium is the basic substance to maintain the survival, growth and proliferation of cells in vitro, and also provides a living environment for the nutrition and reproduction of tissue cells. By improving the culture environment, the amplification factor can be increased in a short period of time, and the differentiation and aging during the amplification process can be reduced. It can be divided into two categories: natural medium and synthetic medium. The nutrients required for culturing cells in vitro are the same as in vivo. The synthetic medium RPMI1640, 199, etc. currently on the market contain enough amino acids, but when using synthetic medium, some natural i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12N5/0775
Inventor 洪敬欣韩俊领刘俊江李茜黄文敬
Owner UNION STEMCELL & GENE ENG
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