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Tissue engineering skin containing appendage and preparation method thereof

A technology for tissue engineering skin and appendages, applied in biochemical equipment and methods, tissue culture, epidermal cells/skin cells, etc., can solve problems such as complex components, prone to rejection, easy to spread diseases, etc.

Active Publication Date: 2017-09-29
GUANGZHOU RAINHOME PHARM&TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The Chinese patent with the notification number CN101773688B discloses a method for preparing tissue-engineered skin containing appendages, which involves inoculating amniotic mesenchymal stem cells, amniotic mesenchymal stem cells, and amniotic epithelial cells induced in the direction of the hair papilla in the gel solution. 1. The amnion epithelial cells induced toward the sweat gland epithelium are cultured in the dermis, and then induced to form hair follicles and sweat gland structures, and then inoculated on the surface with amnion epithelial cells and amnion epithelial cells induced toward keratinocytes. After culture Tissue-engineered skin with hair follicles and sweat gland structures is obtained. 80% volume fraction of the culture medium used in this invention is derived from animal extracellular matrix, which has complex components, is easy to spread diseases, and is prone to rejection reactions

Method used

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  • Tissue engineering skin containing appendage and preparation method thereof
  • Tissue engineering skin containing appendage and preparation method thereof
  • Tissue engineering skin containing appendage and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1 medium composition and kind

[0070] DMEM / F12 mixed with a volume ratio of 9:1 was used as the basal medium, and the following culture medium was prepared using the basal medium

[0071] Culture medium, where the additive concentration is the final concentration:

[0072] Medium B: Mix commercial REGM medium and MEF medium at a volume ratio of 1:1;

[0073] Medium D: basal medium 500ml, L-glutamine 2mmol / L, basic fibroblast growth factor 75ug / L, dexamethasone 10 -8 mol / L;

[0074] Medium E: basal medium 500ml, human epidermal growth factor 20ng / mL, hepatocyte growth factor 3ng / mL, basic fibroblast growth factor 2ng / mL, platelet-derived factor 5ng / mL, insulin-like growth factor 5ng / mL , transforming growth factor-beta 5ng / mL;

[0075] Medium F: basal medium 500ml, keratinocyte growth factor 20ug / L, transforming growth factor-β 45ug / L, platelet-derived factor-AB 8ug / L, vascular endothelial growth factor 15ug / L, interleukin-25ug / L , hydroco...

Embodiment 2

[0082] Embodiment 2 A kind of tissue engineered skin containing appendages

[0083] The preparation method of the tissue-engineered skin is as follows: the medium components involved are as in Example 1, and the steps are:

[0084] Step 1: Collection of urine cells

[0085] 1) After adding 2 mL of penicillin / streptomycin double antibody to the collection cup, collect 120 mL of urine;

[0086] 2) Pour the urine into four 50mL centrifuge tubes, centrifuge at 400r / min for 10min, suck off the supernatant until the remaining liquid in each tube is 3mL, combine them into one centrifuge tube, add 20mL penicillin / strand with a volume fraction of 5% Mycin in PBS, after mixing, centrifuge at 400r / min for 10min, absorb the supernatant until the remaining liquid is 0.75ml;

[0087] 3) Coat 1 well of a six-well plate with 0.1% gelatin for 25 minutes, suck off the liquid in the well, pour the remaining liquid obtained in step 2) into the coated well, add 2 mL REGM medium and 3...

Embodiment 3

[0115] Embodiment 3 A kind of tissue engineered skin containing appendages

[0116] The difference with Example 2 is that the amnion used is decellularized amnion, soaked in DEME medium containing 0.5% mass fraction chitosan for 12 hours before use;

[0117] Step 5, the construction of tissue-engineered double-layer skin containing appendages, as follows:

[0118] The amniotic membrane is punched into 6 amniotic membrane discs with the same hole diameter;

[0119] Mix fibroblasts and hair follicle cells at a ratio of 1.1:1, inoculate into the amnion stroma layer, and the total inoculation density is 2×10 7 / ml, add culture medium I to culture, until the fusion degree of cells reaches 75%, inoculate epidermal cells on the epithelial surface of amniotic membrane, add culture medium G to culture, inoculation density is 2×10 6 / ml;

[0120] After 2 days of culture, the epidermal cells adhered to the amnion epithelium, discarded the culture medium, added 2 mL of...

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Abstract

The invention belongs to the biotechnical field and in particular to tissue engineering skin containing appendage and a preparation method thereof. The preparation method comprises the following steps: performing transfection of a urine cell to induce to generate a multifunctional stem cell; continuously inducing the multifunctional stem cell to generate a mesenchymal stem cell; differentiating the mesenchymal stem cell to form an epidermis cell, a fibroblast and a hair follicle cell; and constructing tissue engineering double-layered skin containing the appendage with a biological stent material amniotic membrane. The seed cell of the tissue engineering skin prepared by the preparation method is the urine cell which is convenient to obtain and short in cultivation period. The hair follicle structure which is contains facilitates union of a wound, and the tissue engineering skin containing the appendage provided by the invention lays a foundation for follow-up development of the tissue engineering skin containing the appendage.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a tissue-engineered skin containing appendages and a preparation method thereof. Background technique [0002] As the largest tissue and organ of the human body, the skin is the first protective barrier of the human body. It can prevent the loss of water and electrolytes in the body, and at the same time protect the body from harmful substances from the outside. The skin is vulnerable to trauma, burns, inflammation, and ulcers , tumor postoperative and congenital diseases and other factors damage. The emergence of tissue-engineered skin provides a new therapeutic approach for research in the field of wound repair. At present, there are many kinds of skin substitutes in the domestic and foreign markets. One is a single-layer tissue-engineered skin with only a dermis layer. This kind of skin substitute has poor ability to isolate external bacteria and is easy to cause infe...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/074C12N5/077A61L27/60A61L27/36A61L27/38
CPCA61L27/3604A61L27/3804A61L27/3886A61L27/60C12N5/0625C12N5/0627C12N5/0656C12N5/0696C12N2501/90C12N2533/92
Inventor 车七石
Owner GUANGZHOU RAINHOME PHARM&TECH CO LTD
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