A kind of serum-free fibroblast 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: 2016-04-27
西安博鸿生物技术有限公司
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  • 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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  • A kind of serum-free fibroblast culture medium and preparation method thereof
  • A kind of serum-free fibroblast culture medium and preparation method thereof
  • A kind of serum-free fibroblast culture medium and preparation method thereof

Examples

Experimental program
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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, bFGF50μg / L, EGF10μg / L, TGF-β10μg / L, PDGF10μg / L, insulin 10mg / L, transferrin 10mg / L; The pH was adjusted to 7.2 with HCI solution, and after constant volume, it was sterilized by filtration with a 0.2 μm filter membrane 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...

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, bFGF10μg / L, EGF5μg / L, TGF-β2μg / L, PDGF2μg / L, insulin-like growth factor 5mg / L, transferrin 5mg / L; use HCI solution to adjust the pH to 7.2, and after constant volume, use a 0.2 μm filter membrane to filter and sterilize 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

A serum-free fibroblast culture medium and a preparation method thereof, the basal medium of the present invention is DMEM medium or its mixing with F12 medium; exogenous additives include hormones, metalloproteins, glutamine, lipids Concentrates, antioxidants, purines, sugars and cell growth factors. Compared with the prior art, the present invention has the following advantages: no animal-derived or recombinant albumin is added, which avoids possible safety hazards and reduces the cost of the medium; it is suitable for the primary and subculture of fibroblasts, and maintains a good The cell morphology is similar to that of the cells in the serum group; it does not need to be coated with adhesion factors, the adhesion rate of the primary 24h is > 60%, and the adhesion rate of the subcultured 24h is > 80%, which is no different from that of the cells in the serum group; It meets the needs of multiple passages, and the growth status remains similar to that of serum group cells after 15 passages; the fibroblasts are directly transferred from the serum-containing culture to the culture of the present invention, and the cells can adhere to the wall and extend smoothly without gradually adapting to the 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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Patent Type & Authority Patents(China)
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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