Non-serum medium suitable for microencapsulation CHO cell and uses thereof

A serum-free medium and microencapsulation technology, applied in tissue culture, animal cells, vertebrate cells, etc., can solve problems such as adverse effects, design of serum-free medium, and complex serum content, reducing the difficulty of separation and purification. cost, improved transplant biosafety, improved biosafety efficacy

Inactive Publication Date: 2008-12-10
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

At present, microencapsulated cells are cultured and transplanted with 10% bovine serum medium for culturing and preservation. The use of serum has the following disadvantages: 1) it contains potential cytotoxicity and the risk of animal-derived contamination. Microencapsulated cells adverse effects during transplantation
2) The content of serum is very complex, increasing the difficulty and cost of downstream separation and purification of biological products
3) Differences between serum batches increase the instability of microencapsulated cell products
4) The protein contained in the serum may cause microcapsule membrane pollution during microencapsulated cell culture and affect material transport, resulting in the death of cells in the capsule due to nutrient imbalance
[0003] Although there are many types of commercially available medium (document 2. A serum-free medium suitable for the cultivation of Chinese hamster ovary cells. Application No. 200410018258.0), there is no special serum-free medium designed for the metabolic characteristics of microencapsulated cells

Method used

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  • Non-serum medium suitable for microencapsulation CHO cell and uses thereof
  • Non-serum medium suitable for microencapsulation CHO cell and uses thereof
  • Non-serum medium suitable for microencapsulation CHO cell and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] ① The culture medium is prepared according to the following formula

[0030] The culture medium among the present invention is a mixture, is made up of commercialized DMEM / F12 standard culture medium (purchased from Sigma Company, specific composition sees Table 1) and following supplement:

[0031] Insulin 15mg / L

[0032] Soy Lecithin 40mg / L

[0033] Tween 80 20mg / L

[0034] Tropolone 1μM

[0035] Ferric ammonia (FAC) 0.1mg / L

[0036] Sodium selenite 2.5mg / L

[0037] β-Mercaptoethanol 20μM

[0038] L-Aspartic Acid 1mg / L

[0039] L-Glycine 8mg / L

[0040] L-Arginine 5mg / L

[0041] L-proline 1mg / L

[0042] L-histidine 4mg / L

[0043] L-Lysine 3mg / L

[0044] L-cysteine ​​10mg / L

[0045] L-Glutamine 100mg / L

[0046] All of the above substances are analytically pure chemical reagents.

[0047] The culture medium of the present invention is prepared by a conventional mixing method.

[0048] The culture medium described in the present invention can be used accord...

Embodiment 2

[0056] ① The culture medium is prepared according to the following formula

[0057] Add the following supplements to 1L DMEM / F12 medium, and then add this medium to the culture flask, the cell density of CHO cells after microencapsulation is 2×10 6 cells / ml, cultivated for 7 days.

[0058] Insulin 5mg / L

[0059] Soy Lecithin 10mg / L

[0060] Tween 80 20mg / L

[0061] Tropolone 3μM

[0062] Ferric ammonia (FAC) 1mg / L

[0063] Sodium Selenite 10mg / L

[0064] β-Mercaptoethanol 50μM

[0065] L-Aspartic Acid 5mg / L

[0066] L-Glycine 20mg / L

[0067] L-Arginine 10mg / L

[0068] L-Proline 5mg / L

[0069] L-histidine 10mg / L

[0070] L-Lysine 8mg / L

[0071] L-cysteine ​​20mg / L

[0072] L-Glutamine 400mg / L

[0073] 2. Preparation of microencapsulated CHO cells (same as Example 1)

[0074] ③ microencapsulated cells at 37°C, 5% CO 2 The cell density after 7 days in the incubator was 16×10 6 cells / ml microcapsules.

Embodiment 3

[0076] ① The culture medium is prepared according to the following formula

[0077] Add the following supplements to 1L DMEM / F12 medium, and then add this medium to the culture flask, the cell density of CHO cells after microencapsulation is 2×10 6 cells / ml, cultivated for 7 days.

[0078] Insulin 10mg / L

[0079] Soy Lecithin 30mg / L

[0080] Tween 80 40mg / L

[0081] Tropolone 5μM

[0082] Ferric Ammonia (FAC) 0.5mg / L

[0083] Sodium selenite 8mg / L

[0084] β-Mercaptoethanol 30μM

[0085] L-Aspartic Acid 3mg / L

[0086] L-Glycine 3mg / L

[0087] L-Arginine 1mg / L

[0088] L-proline 10mg / L

[0089] L-histidine 1mg / L

[0090] L-Lysine 10mg / L

[0091] L-cysteine ​​3mg / L

[0092] L-Glutamine 200mg / L

[0093] 2. Preparation of microencapsulated CHO cells (same as Example 1)

[0094] ③ microencapsulated cells at 37°C, 5% CO 2 The cell density after 7 days in the incubator was 15×10 6 cells / ml microcapsules.

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Abstract

The invention relates to cell culture and transplantation, in particular to a serum-free medium which is suitable for microencapsulated CHO cells, wherein, DMEM/F12 is taken as a basic medium; the characteristics of growth and metabolism of the microencapsulated cells are focused; and a certain proportion of insulin, soybean lecithin, Tween 80, Tropolone, FAC, sodium selenite, beta-mercaptoethanol and amino acid are added. The serum-free medium can promote vigorous growth of the microencapsulated CHO cells, and the cell density, the product expression and the activity all reach or are superior to the cell culture level of a serum medium, thereby the serum-free medium can replace the serum medium to be used for culture and transplantation of the microencapsulated CHO cells, greatly reduce the culture cost, and improve the biological safety of the microencapsulated animal cells.

Description

technical field [0001] The invention relates to cell culture and transplantation, in particular to a serum-free medium suitable for microencapsulating Chinese hamster ovary (CHO) cells and application thereof. Background technique [0002] Microencapsulated cell culture and transplantation is an artificial organ and drug release technology with broad application prospects, which can be applied to the treatment of many diseases, including: diabetes, liver failure, tumors, etc. This technique was proposed in 1964 by TMS Chang (Document 1. Chang T M S, 1964. Semipermeable microcapsules. Science 146, 524-525). Because the microcapsule membrane has selective permeability and immune isolation, the use of immunosuppressants in the process of cell transplantation is reduced. In addition, microencapsulation technology has begun to be used in large-scale cell culture, monoclonal antibody production and other bioengineering fields. At present, microencapsulated cells are cultured and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N11/04C12N5/071
Inventor 马小军吕国军李娜王为林军章于炜婷孙志杰
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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