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Preparation method and application of autologous fibroblasts

A fibroblast and autologous technology, applied in the field of fibroblasts, can solve the problems of large cell damage, low preparation and separation efficiency, complicated preparation process, etc., and achieves high cell safety, low skin extraction area requirements, and high preparation efficiency. Effect

Inactive Publication Date: 2018-09-04
BEIJING DOING TIMES BIOMEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Autologous fibroblasts are obtained from their own skin cells. After in vitro expansion, these living cells are injected into the cortex to further produce collagen, increase the density and thickness of collagen in the dermis, restore skin elasticity, and allow it to grow out of skin tissue Make the grooves flat to achieve the purpose of eliminating wrinkles and scars; at present, the preparation of autologous skin fibroblasts is mainly prepared by enzymatic digestion, but this method needs to provide enough skin tissue, so it is not suitable for small-area skin tissue isolation cells. Moreover, the preparation process of the enzymatic digestion method is cumbersome, especially the separation of the dermis and epidermis requires a 4°C digestion liquid, and the enzymatic digestion is relatively harmful to the cells, and the preparation and separation efficiency is low.

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  • Preparation method and application of autologous fibroblasts
  • Preparation method and application of autologous fibroblasts
  • Preparation method and application of autologous fibroblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Human autologous fibroblasts are isolated and expanded and cultivated

[0042] (1) Isolation of human autologous fibroblasts

[0043] 1) Under a sterile environment, use a sterile skin biopsy puncher to take 3mm skin tissue behind the ear;

[0044] 2) Remove the skin and immediately transfer it to a biological safety cabinet, and rinse it with physiological saline (containing 2% penicillin and streptomycin mixed solution) for 3-5 times;

[0045] 3) Under a stereo microscope, use micro tweezers and a surgical blade to evenly cut 6-9 small pieces of the cleaned skin tissue;

[0046] 4) Evenly spread the cut skin tissue into a 3.5 cm culture dish coated overnight with 1 ml of 0.5% Matrigel (manufacturer: BD; product number: 356234) with an interval of 2 cm between the tissue pieces; add 400 ul of fibroblast culture medium ( Manufacturer: PromoCell GmbH; Item No.: C-23010) placed at 37°C, 5% CO 2 Primary cell culture was carried out in the incubator, and 20...

Embodiment 2

[0049] Embodiment 2: autologous fibroblast identification

[0050] (1) Viability detection of autologous fibroblasts

[0051] Separate the P0 generation autologous fibroblasts in Example 1 to make a single cell suspension, draw 20 μl of the upper cell suspension into a test tube, add 20 μl of 0.4% trypan blue, mix well, and count with a cell counting board after 3 minutes; Observed under the microscope, the dead cells were light blue, and the live cells were rejected, and counted under a 200×high magnification microscope; data analysis, cell survival rate=(total cell number-dead cell number) / total cell number×100%.

[0052] Results: In this experiment, P0 generation autologous fibroblasts were taken for trypan blue exclusion test for three consecutive times, and the viable cell rates of the three checks were 98%, 99%, and 99% respectively.

[0053] (2) Growth curve of autologous fibroblasts

[0054] 1) Take the P5 autologous fibroblasts amplified in Example 1, digest them wi...

Embodiment 3

[0078] Embodiment 3: Determination of collagen content (hydroxyproline) in autologous fibroblast culture fluid

[0079] Since the collagen synthesized by the cultured cells is directly secreted into the culture medium, the content of hydroxyproline (HYP) in the culture medium can indirectly reflect the amount of collagen synthesized by the cultured cells. The culture supernatants of autologous fibroblasts were collected at different time points, and the HYP content was determined with a hydroxyproline test kit.

[0080] For the operation steps, refer to the instructions of Hydroxyproline Detection Kit (manufacturer: Nanjing Jiancheng Bioengineering Research Institute; article number: A030-1):

[0081]

blank tube

standard tube

Assay tube

Double distilled water (ml)

0.25

5ug / ml standard application solution

0.25

Fibroblast culture supernatant (ml)

0.25

Digestive fluid (ml)

0.05

0.05

0.05 ...

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Abstract

The invention discloses a preparation method and application of autologous fibroblasts. The preparation method of the autologous fibroblasts comprises the following steps that autologous skin tissue of (2.5-3.5 mm)*(2.5-3.5mm) is taken, washed and cut; the cut autologous skin tissue is evenly flatly laid in a culture dish, a fibroblast culture medium is added, and the autologous skin tissue is placed in a culture box with the temperature being 37 DEG C and the CO2 concentration being 5% for primary cell culture; and after the cell confluence degree reaches 70%-80%, trypsin is used for removingepithelial cells in a digestion mode, then digestion is terminated through the fibroblast culture medium, subculture is conducted, and the autologous fibroblasts are obtained. The autologous fibroblasts prepared through the method are high in purity, high in safety and excellent in performance.

Description

technical field [0001] The invention relates to the field of fibroblasts, in particular to a preparation method and application of autologous fibroblasts. Background technique [0002] Fibroblasts are the most important cells in the dermis, often attached to collagen fibers, and can synthesize and secrete a large amount of collagen and elastin. Under normal circumstances, they are in a relatively static state, and can enter proliferation when the skin is damaged. It participates in tissue repair and is the main repair cell after the skin tissue is damaged. It forms the main body of the dermis together with the collagen fibers, elastic fibers and matrix components secreted by itself. It is an important substance related to the quality and state of the skin. Smoothness, luster, elasticity, and toughness are all inseparable from it. [0003] Autologous fibroblasts are taken from their own skin cells. After in vitro expansion, these living cells are injected into the cortex to ...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/071A61K8/99A61K8/98A61Q19/00
CPCA61K8/981A61K8/99A61Q19/00C12N5/0656C12N2506/09
Inventor 穆小生徐珊珊董飞
Owner BEIJING DOING TIMES BIOMEDICAL TECH
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