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Serum-free fibroblast cell culture medium and preparation method thereof

A fibroblast and culture medium technology, applied in the field of biomedical materials, can solve the problems of increasing operation complexity, increasing the cost of culture medium, and high cost of culture medium, and achieves the advantages of large-scale culture, vigorous cell proliferation, and good stretching Effect

Active Publication Date: 2014-07-09
西安博鸿生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are common problems in serum-free media used for other cell cultures: ①As the main component of serum, albumin is a common additive in serum-free media. Since bovine serum albumin (BSA) is an animal-derived protein , may carry pathogenic factors; the current animal-free medium mostly uses recombinant albumin (HSA) instead of BSA, resulting in higher cost of the medium
② Serum contains a variety of adhesion factors, and cells that depend on serum are not easy to adhere to the wall when cultured without serum. Therefore, when culturing adherent cells without serum, they need to be coated with adhesion factors in advance, which increases the complexity of the operation, and Commonly used anchorage factors (such as collagen, polylysine, fibronectin, laminin, etc.) are expensive, increasing the cost of the medium

Method used

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  • Serum-free fibroblast cell culture medium and preparation method thereof
  • Serum-free fibroblast cell culture medium and preparation method thereof
  • Serum-free fibroblast cell culture medium and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, primary culture of human fibroblast

[0035] Prepare medium: mix DMEM medium and F12 medium at a volume ratio of 3:1; add dextran at 30 g / L and stir to dissolve, then add triiodothyronine 5 μM and hydrocortisone 5 mg / L respectively , dexamethasone 5μg / L, adenine 20mg / L, sodium selenite 15μg / L, 2-mercaptoethanol 50μM, V E 5mg / L, V C 50mg / L, lipid concentrate 1mL / L, glutamine 5mM, ethanolamine 5μM, bFGF 50μg / L, EGF 10μg / L, TGF-β 10μg / L, PDGF 10μg / L, insulin 10mg / L, transferrin 10mg / L; use HCI solution to adjust the pH to 7.2, and after constant volume, use a 0.2μm filter membrane to filter and sterilize to obtain a serum-free fibroblast culture medium.

[0036] Example of use: wash the digested human fibroblasts with PBS solution; resuspend with the prepared serum-free fibroblast medium after centrifugation, press 2×10 4 piece / cm 2 Inoculate into a culture bottle and place in an incubator at 37°C, 5% CO 2 Culture under conditions; change the medium ...

Embodiment 2

[0038] Embodiment 2, subculture of human fibroblast

[0039] Prepare medium: take DMEM medium as the base medium, add dextran at 10 g / L and stir to dissolve, then add triiodothyronine 1 μM, hydrocortisone 0.05 mg / L, and dexamethasone 0.05 μg / L respectively. L, adenine 30mg / L, sodium selenite 2μg / L, 2-mercaptoethanol 100μM, V E 5mg / L, V C 10mg / L, lipid concentrate 1mL / L, glutamine 2mM, ethanolamine 20μM, bFGF 10μg / L, EGF 5μg / L, TGF-β 2μg / L, PDGF 2μg / L, insulin-like growth factor 5mg / L, Transferrin 5 mg / L; adjust the pH to 7.2 with HCI solution, and filter to sterilize with a 0.2 μm filter membrane after constant volume to obtain a serum-free fibroblast culture medium.

example 1

[0040] Example 1: Digest the cells that were primary cultured to 60% confluence in Example 1, wash them with PBS solution, centrifuge and discard the supernatant, and resuspend the cells with the serum-free medium prepared in this example, press 1×10 4 piece / cm 2 The cell density was seeded at 37°C, 5% CO 2 The conditional incubator was used for culture; the medium was changed every 3 days, and the next passage could be carried out when the cells grew confluent to 80%.

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Abstract

The invention relates to a serum-free fibroblast cell culture medium and a preparation method thereof, wherein the base culture medium is a DMEM culture medium or a mixture of the DMEM culture medium and a F12 culture medium, and the exogenous additives comprise hormones, a metal transporter, glutamine, a lipid concentration agent, an antioxidant, purine, saccharide and a cell growth factor. Compared with the culture medium in the prior art, the serum-free fibroblast cell culture medium of the present invention has the following advantages that: animal origin or recombinant albumin is not added so as to avoid the possibly-carried safe hidden danger and reduce the culture medium cost; the culture medium is suitable for primary culture and subculture of fibroblast cells, the good cell morphology is maintained, and the status and the function of the cultured cells are similar to the status and the function of the serum group cells; adherence factor coating is not required, the primary 24 h adherence rate is more than 60%, the passage 24 h adherence rate is more than 80%, no difference exists between the cells cultured with the culture medium of the present invention and the serum group cells; requirements of multiple passages can met, and the growth conditions of the cells after 15 passages are similar to the growth conditions of the serum group cells; and after the fibroblast cells are directly transferred into the culture of the present invention from the serum-containing culture, the cells can successfully adhere and extend without the gradual adaptation process.

Description

technical field [0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a serum-free fibroblast culture medium and a preparation method thereof. Background technique [0002] Tissue-engineered skin is currently a hot spot in the field of tissue engineering, and many products have been used clinically in recent years. A major problem to be solved in the research and development of tissue engineered skin is the large-scale in vitro culture of seed cells (ie, epidermal cells and fibroblasts). [0003] Traditional fibroblast culture is to add serum to the basal medium, but the use of serum will bring risks and hidden dangers: ① Serum contains cytostatic factors and toxic factors, which have potential cytotoxicity; ② Serum components are complex, batches There are differences in the quality of the product, which increases the difficulty and instability of product quality control; ③Currently, the mechanism of action of each compon...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 贺晓静
Owner 西安博鸿生物技术有限公司
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