Non-animal source cell blood serum substitute and use thereof

A serum substitute and cell culture technology, which is applied in the field of non-animal-derived cell culture serum substitutes, can solve problems such as the inability to meet the demand for the number of cells in clinical cell therapy, the inability to achieve rapid cell expansion, and the potential safety hazards of clinical treatment. , to achieve the effect of shortening the operation cycle, avoiding high risks, and meeting clinical safety

Active Publication Date: 2009-07-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the cell therapy of regenerative medicine, there are not only quantitative requirements for the seed cells used clinically, but also the need to ensure their safety and effectiveness. Although the participation of animal-derived serum in cell culture can meet ...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: PLT acquisition method specific operation steps

[0017] (1) Collect whole blood (450ml±45ml) into a blood bag (citrate phosphate dextrose blood bag);

[0018] (2) After standing at 22°C for 16 hours, the blood was centrifuged (4250g, 13min, 22°C) to separate red blood cells and platelets to obtain buffy coat and plasma. Instrumentation (CompomatG3, NPBI, Amsterdam, the Netherlands));

[0019] (3) To avoid agglutination reaction caused by MSC; four units of O-type buffy coat mixed with one unit of AB plasma, and gently centrifuged (341g, 6min, 22°C) as one unit of platelet-rich plasma (PRP). The PRP was transferred into a storage bag and filtered to remove leukocytes. Freeze at -30°C. Before use, thaw at 37°C and centrifuge (4000g, 15min) to remove PLT membrane fragments and reduce the risk of triggering an immune response between allotypes.

[0020] (4) The supernatant is PLT.

Embodiment 2

[0021] Example 2: PLT as a serum substitute for culturing mesenchymal stem cells

[0022] Medium preparation: Add 50mL PLT, 1000U heparin, 1mmol glutamine, 5mL double antibody (10000U / mL for penicillin and streptomycin respectively) to 500ml DMEM basal medium; primary cell culture:

[0023] 1. Extract 5ml of bone marrow and anticoagulate with heparin; use PLT medium for differential adherence culture;

[0024] 2. Change the liquid for the first time after 36 hours, 75cm 2 Add 15ml of medium to the cell culture flask.

[0025] 3. Change the medium every 2 to 3 days; trypsinize and count on the 5th to 6th day, this is the primary cell acquisition process; cell expansion:

[0026] 1. Initially use 3×10 5 Cells were planted, and the medium was changed every 2 days, and the cell density reached more than 90%.

[0027] 2. Trypsin digestion, each bottle with 3 × 10 5 Plant at a cell density, change the medium once every 2-3 days;

[0028] 3. After 11-14 days of amplification an...

Embodiment 3

[0030] Example 3: PLT as a serum substitute for culturing human chondrocytes

[0031] Medium preparation: add 50mL PLT, 1000U heparin, 1mmol glutamine, 5mL double antibody (10000U / mL for penicillin and streptomycin respectively) to 500ml DMEM basal medium; use 75cm 2 Cell expansion in culture flasks;

[0032] Cell Expansion:

[0033] 1. Take 1×10 6 Cells / bottle were planted, the medium was changed every 2 days, and the cell density reached more than 90% for passage.

[0034] 2. Trypsinized and subcultured, the planting density was 1.5×10 6 / bottle; change the liquid every 2-3 days;

[0035] 3. After 11-14 days of amplification and subculture, a total of not less than 4.5×10 8 cells. Ectopic Chondrogenic Culture of Chondrocytes:

[0036] 1. Collect 10 6 More than chondrocytes;

[0037] 2. Suspend with 0.25ml collagen gel and 0.25ml cartilage matrix to form a gel mass;

[0038] 3. The agglomerate was implanted subcutaneously in the back of nude mice, and it was taken o...

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PUM

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Abstract

The invention provides a non-animal origin cell cultivating serum substitute (PLT) and applications thereof. The PLT is prepared by the following method: 3 to 5 volume units of O-typed buffy coats and 1 volume unit of AB-typed plasma are mixed; 300 to 400 grams of the mixture is centrifuged; a supernatant is extracted as 1 volume unit of platelet-rich plasma; the platelet-rich plasma is filtered so as to remove leucocyte, stored under cryopreservation at the temperature below minus 10 DEG C for standby, and unfrozen at the temperature of 30 to 37 DEG C before use, and 3000 to 5000 grams of the platelet-rich plasma is centrifuged so as to remove platelet membrane fragments, thus obtaining the non-animal origin cell cultivating serum substitute. The non-animal origin cell cultivating serum substitute has following essential advantages: the PLT can be used as fetal bovine serum substitute in cell cultivation, thus avoiding animal-origin ingredients in cell cultivation, quickening cell multiplication and consequently satisfying the requirements of clinical security and operation period shortening, and the non-animal origin cell cultivating serum substitute is applicable not only to mesenchymal stem cells, but also to the multiplication cultivation of other human origin cells.

Description

(1) Technical field [0001] The invention relates to a non-animal source cell culture serum substitute and its application in cell culture as a substitute for fetal bovine serum and the like. (2) Background technology [0002] In conventional cell culture and expansion processes, animal-derived serum such as fetal bovine serum (FBS) is an essential additive for cell expansion. [0003] In the cell therapy of regenerative medicine, there are not only quantitative requirements for the seed cells used in clinical practice, but also the need to ensure their safety and effectiveness. Although the participation of animal-derived serum in cell culture can meet the quantitative requirements, it brings unsafe hidden dangers to clinical treatment. However, autologous serum is inconvenient to obtain and use, and cannot achieve the effect of rapid cell expansion, nor can it meet the needs of clinical cell therapy for the number of cells. (3) Contents of the invention [0004] The obje...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/071C12N5/077C12N5/0775
Inventor 姜洋子欧阳宏伟
Owner ZHEJIANG UNIV
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