Non serum substratum for in vitro culture and amplification of cutaneous keratin cell

A serum-free medium and skin keratin technology, applied in the field of serum-free medium and medium, can solve the problems of accelerated keratinocyte differentiation, growth and differentiation interference, not serum-free medium for keratinocytes, etc. Proliferative effect

Inactive Publication Date: 2005-01-05
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, keratinocytes cultured with these culture systems and media have various problems in basic research and clinical application: adding serum to the culture medium, on the one hand, causes contamination of fibroblasts, affects the growth of keratinocytes, and accelerates keratinocyte differentiation; On the one hand, due to the existence of serum and proteins with unknown components, it interferes with basic research such as cell biology, toxicology, and pathology (such as: studying the effects of cytokines and drugs on the growth and differentiation of keratinocytes, etc.); In vivo transplantation of artificial skin tissue constructed with medium may cause immune reactions or introduce unsafe factors (such as viruses or other pathogens, etc.), so it is necessary to develop a serum-free medium with defined chemical components
Following MCDB152, literature [Boyce, S.C., Ham, R.G., J.Invest.Dermatol.81, pp.33S-40S(1983); J.Tiss.Cult.Meth.9, pp.83-93(1985)] It was disclosed that the calcium ion concentration was further improved by Boyce and Ham, and it was officially named as MCDB153 medium, but MCDB153 is not an excellent serum-free medium for keratinocytes, and its limitations are limited in the ability to reproduce and expand cells. Keratinocytes will rapidly lose their ability to be passaged if grown to full confluence in MCDB153
[0007] Due to serious deficiencies in the composition of serum-free media, when using these media to culture keratinocytes, dialyzed serum, pituitary extract, cholera toxin, or growth substrates have to be added, making it difficult to truly achieve keratinocyte growth. Serum-free culture in vitro, the cells are prone to aging during the culture process, which affects the massive expansion of cells

Method used

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  • Non serum substratum for in vitro culture and amplification of cutaneous keratin cell
  • Non serum substratum for in vitro culture and amplification of cutaneous keratin cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Using MCDB153 as the base medium, its components are as follows: (in 1L)

[0055] MCDB153 17.61g

[0056] Histidine 0.3mmol

[0057] Isoleucine 0.6mmol

[0058] Tryptophan 0.2mmol

[0059] Phenylalanine 0.3mmol

[0060] Lysine 0.7mmol

[0061] Methionine 0.2mmol

[0062] Tyrosine 0.3mmol

[0063] Calcium chloride 0.1mmol

[0064] Magnesium chloride 5mmol

[0065] Keratinocyte Growth Factor 100ng

[0066] Pantothenate 0.021mmol

[0067] Choline chloride 0.171mmol

[0068] Folic acid 0.024mmol

[0069] Inositol 0.211mmol

[0070] Niacinamide 0.082mmol

[0071] Vitamin B6 0.049mmol

[0072] Riboflavin 0.003mmol

[0073] Thiamine 0.03mmol

[0074] Bovine insulin 5μg

[0075] Transferrin 10μg

[0076] Ethanolamine 0.05mmol

[0077] Strontium chloride 1mmol

[0078] Bovine serum albumin 10μg

[0079] Hydrocortisone 0.1μg

[0080] Triiodothyronine 40pmol

[0081] Mercaptoethanol 0.05mmol

[0082] Penicillin: 100U

[0083] Streptomycin: 100U

[0084] D...

Embodiment 2

[0086] Take the skin of newborn SD rats or human foreskin, cut into 1cm 2 The left and right skin blocks were washed 3 times with PBS containing 200 U / mL gentamycin, 2 times with 75% alcohol, and 2 times with PBS. Add the skin tissue to 200U / mL neutral protease, digest at 4°C for 18 hours, separate the dermis from the epidermis, then cut the epidermis into small pieces, act with 6 mL of 0.025% trypsin / 0.02% EDTA for 5 min, and centrifuge at 2000 rpm for 5 min. And add 10mg / mL soybean trypsin inhibitor 2-3mL to neutralize trypsin activity. Filter through a 150-mesh sieve to remove skin fragments. Centrifuge at 2000 rpm for 5 min, discard the supernatant, wash with PBS twice, and add the keratinocyte serum-free medium of Example 1. Cell seeding volume was 2×10 4 cells / cm 2 , each 25cm 2 Add 5mL of culture medium to the culture flask, at 37°C, 5% CO 2 cultured in an incubator, changing the medium every three days. The keratinocytes can be subcultured after 7-10 days to cov...

Embodiment 3

[0088] Add 3 mL of 0.025% trypsin / 0.02% EDTA to the primary cultured keratinocytes for digestion, centrifuge at 2000 rpm for 5 min after the cells become round and fall off, discard the digestion solution, and add 2-3 mL of 10 mg / mL soybean trypsin inhibitor to neutralize the trypsin enzyme activity. After centrifuging at 2000rpm for 5min, discard the supernatant, wash twice with PBS, and wash with 2×10 4 The cells / mL inoculum was inoculated, and 5 mL of the serum-free medium involved in the present invention was added to each culture bottle for subculture. attached figure 2 The growth curves of mouse keratinocytes in the serum-free medium of the present invention and the traditional medium were compared. The serum-free medium of the present invention adopts the medium of Example 1.

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Abstract

The invention discloses a serum-free culture medium used for culturing and amplifying skin keratinocytes in vitro. It adds amino acid, vitamin, hormone, growth gene, trace elements, and antioxidant, thus able to accelerate cell breeding and delay consenescence of the keratinocytes and makes them amplified in vitro by more times. Using the culture medium to make primary culture and subculture needs no trophoblast cells and no extracellular substrates like collagen, etc prepaved at the bottom of the culture bottle to help the keratinocytes adhere to the wall and grow.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a culture medium, in particular to a serum-free culture medium with defined chemical composition for in vitro culture and expansion of epidermal keratinocytes isolated from skin tissue. Background technique [0002] Patients with skin burns and other skin diseases were previously treated with autologous skin grafts or cadaveric skin grafts, and the source of skin was very limited. With the development of biotechnology, tissue engineering methods can be used to construct artificial skin in vitro, so as to solve the problem of skin origin. [0003] The construction of skin tissue requires a large number of seed cells, and the in vitro culture of epidermal keratinocytes is a difficult point. Keratinocytes are a kind of committed stem cells, which are easy to age and differentiate during in vitro culture, thereby affecting cell proliferation; secondly, due to changes in the growth environ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/08
Inventor 谭文松周燕朱宏庆华平
Owner EAST CHINA UNIV OF SCI & TECH
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