Stem cell complex for inducing hair regeneration as well as preparation method and application of stem cell complex

A technology of stem cells and complexes, applied in the field of stem cells, can solve the problems of lack of specific cell marker molecules and low screening efficiency, and achieve the effect of avoiding complicated and cumbersome transplantation techniques, avoiding insufficient donor sites, and promoting directed differentiation

Inactive Publication Date: 2018-07-27
朱剑虹
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although undifferentiated cells can be removed by flow cytometry, there is still a lack of very specific cell marker molecules, and the screening efficiency is not high

Method used

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  • Stem cell complex for inducing hair regeneration as well as preparation method and application of stem cell complex
  • Stem cell complex for inducing hair regeneration as well as preparation method and application of stem cell complex
  • Stem cell complex for inducing hair regeneration as well as preparation method and application of stem cell complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 Preparation and application of a stem cell complex for inducing hair regeneration, comprising the following steps:

[0049] 1) Fabrication of cell culture scaffolds

[0050] Use αMEM matrix, buffer, and acidic collagen to prepare collagen gel mixture: 100 μL ten-fold concentrated αMEM matrix, 100 μL buffer (NaOH 0.08M, HEPES 200mM), 800 μL acidic collagen solution (3mg / ml , pH3.0).

[0051] Add the above mixed solution into a sterile EP tube, use a sterile pipette tip to blow up and down to mix, use the pipette tip to suck 30-40μl and drop it on a dish or 24-well plate, and centrifuge at 300g for 10 minutes at 4°C to prevent condensation Bubbles were formed in the gel, and then the plate was incubated at 37°C for 1 hour to promote the gel to form a colloidal hanging drop.

[0052] The biomechanical range of the obtained collagen gel hanging drop: the initial elastic modulus is 13-18KPa, the maximum tensile stress is 100-500KPa, the stress relaxation time ...

Embodiment 2

[0064] Except that the steps of preparing and assembling stem cells in step 2) are different from those in Example 1, the rest of the operations are the same as in Example 1.

[0065] The steps of preparing and assembling stem cells in this example are as follows:

[0066] Recover cryopreserved HiPSCs.

[0067] First, trypsin was warmed in a 37°C water bath, and the recovered HiPSCs were washed twice with PBS, added with trypsin (0.25% trypsin: 0.4% EDTA = 1:1), and then placed at 37°C, CO 2 Incubate in an incubator with a saturated humidity of 5% for 1min, add 1-2ml of fetal bovine serum to stop trypsin, scrape the bottom of the culture plate with a pipette and blow the clones, then transfer the cell suspension to a 10ml conical centrifuge tube, Break up the colonies at the bottom of the centrifuge tube until no cell clumps are visible and a relatively homogeneous cell suspension is formed.

[0068] Centrifuge at 1400r / min for 5min, remove the supernatant, resuspend the cel...

Embodiment 3

[0075] Except step 2), all the other operations are with embodiment 2.

[0076] Prepare and assemble stem cells as follows:

[0077] After recovery from cryopreserved HiPSCs, inoculate 90 HiPSCs / 30 μl into a Nunclon round-bottom 96-well plate, add 30 μl of 15% fetal bovine serum DMEM medium to each well plate, and cultivate embryoid bodies on the third day , and continued to culture until the sixth day to obtain embryoid bodies.

[0078] Among them, in the culture medium, add TGF-β2 50ng / ml on day1; add RA1ng / ml on day2, and add RA 1ng / ml, TGF-β2 50ng / ml, EGF 20ng / ml, adenosine 30mg / ml after replacing the fresh medium on day3 , day 6 days after replacement of fresh medium, add Wint 10b 500ng / ml, Wint 3a 100ng / ml, adenosine 30mg / ml, PFI-3 2mmol / L.

[0079] Prepare siliconized-coated 35-mm Petri dishes and 1.5-mL silicone-coated test tubes, place 30 μL of collagen hanging drops on 35-mm silicone-coated tissue culture dishes, and transfer embryoid body units to In the test tube,...

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Abstract

The invention discloses a stem cell complex for inducing hair regeneration as well as a preparation method and an application of the stem cell complex. The stem cell complex comprises a cell culture scaffold and stem cells, wherein a main body of the cell culture scaffold is a collagen gel hanging drop, and the stem cells are placed in the collagen gel hanging drop; the stem cell complex also comprises an absorbable suture with one end extending out of the surface of collagen gel. In the stem cell complex, the stem cells can be differentiated into a precursor stem cell line similar to organ tissue directionally under the induced differentiation condition, the method simulates the process of the interaction between epithelial cells and mesenchymal cells for hair follicle development, hair with complete functions can be generated after transplantation, and transplantation of the stem cells is more similar to that of tissue organs.

Description

technical field [0001] The invention belongs to the field of stem cells, and in particular relates to a stem cell complex for inducing hair regeneration, its preparation method and application. Background technique [0002] The presence of hair is a hallmark of mammals and serves a wide range of roles, including physical protection, insulation, cover, dispersion of sweat and fat, sensory and tactile functions, and social interactions. [0003] The average total number of healthy adult hairs is 50,000 to 150,000, and its growth is affected by various factors such as age, climate, environment, and health status, but the main structure that regulates the periodic growth of hair is the hair follicle cell. Hair follicles regenerate themselves periodically and regularly, and continue throughout life in the human body. However, alopecia can be caused due to androgen degeneration or mutation, alopecia areata, seborrheic skin, trauma, etc., which means that the hair on the long hair...

Claims

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Application Information

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IPC IPC(8): A61L27/38A61L27/24A61L27/58A61L27/60
CPCA61L27/24A61L27/3834A61L27/3886A61L27/58A61L27/60A61L2430/18
Inventor 魏乃礼朱剑虹王帆朱侗明
Owner 朱剑虹
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