Method for constructing 3D pseudo-epidermis based on organ chip and directed differentiation of induced pluripotent stem cells

A technology of pluripotent stem cells and organ chips, applied in the field of 3D pseudo-epidermal construction, can solve the problems of unsatisfactory, insufficient skin source materials, inability to realize multi-cell co-cultivation and drug screening, etc., to promote cell adhesion, good microscopic environmental effects

Active Publication Date: 2021-11-09
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the main problem of autologous transplantation is that the skin source material is insufficient, which cannot meet the situation of large-scale skin damage. Allogeneic / xenograft transplantation will bring certain immune rejection. Flaws prevent co-cultivation and drug screening of multiple cell types

Method used

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  • Method for constructing 3D pseudo-epidermis based on organ chip and directed differentiation of induced pluripotent stem cells
  • Method for constructing 3D pseudo-epidermis based on organ chip and directed differentiation of induced pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Inoculation of iPSCs

[0049] When iPSC reaches the passage state, it can be used for inoculation. The specific steps are as follows:

[0050] Take out the PSCeasy human pluripotent stem cell (ES / iPS) medium (iPSC medium for short) and put it at room temperature, take out the culture dish coated with Matrigel Matrix (Matrigel, Matrigel for short, from Corning Company in the United States), and suck Remove the coating solution and add an appropriate amount of iPSC medium, place in 5% CO 2 In a 37°C constant temperature cell incubator, 37°C water bath preheated PBS solution (phosphate buffered solution, referred to as PBS, from Gibco in the United States) and 0.5mM EDTA passaging working solution (ethylenediaminetetraacetic acid, referred to as EDTA, from Thermo Fisher Corporation of the United States).

[0051] The old iPSC medium was aspirated, and apoptotic iPSCs were removed by rinsing the bottom of the dish by adding calcium and magnesium-free PBS soluti...

Embodiment 2

[0058] Example 2: Differentiation of iPSCs into keratinocytes

[0059] On Day0, on the second day after the iPSCs with an inoculation density of about 30% adhered to the wall, 2 ml of Definedkeratinocyte serum-free medium (keratinocyte serum-free medium, referred to as DKSFM, derived from Gibco in the United States) medium (1um all -trans RA (retinoic acid, referred to as RA, derived from Sigma Company in the United States), 10ng / ml Recombinant Human BMP-4 Protein (bone morphogenic protein, referred to as BMP-4, derived from R&D Systems Company in the United States) and 1.5mm CaCl 2 ), change the medium every two days, 37℃, 5%CO 2 Incubator cultivation.

[0060] On Day 6, the old medium was aspirated, and 2 ml of Defined keratinocyte serum-free medium (DKSFM) medium was added. The DKSFM medium did not contain RA, BMP4 and CaCl2, and was replaced every two days until keratinocytes were initially differentiated.

[0061] On Day 16, the old medium was aspirated, and an appropri...

Embodiment 3

[0062] Example 3: Differentiation of iPSCs into fibroblasts

[0063] Preparation of embryoid bodies (EBs): first to Aggrewell TM 400 (microwell Petri dish, referred to as Aggrewell TM 400, from Canada's STEMCELL company), add DMEM medium, rinse and suck out. Join Aggrewell TM medium, centrifuge at 1000rmp for 5min, remove air bubbles, digest iPSCs with a density of 80-90% adherent with 0.5mM EDTA, pipette the cells with PBS and centrifuge, and aspirate the supernatant.

[0064] After that, remove the spare Aggrewell from the incubator TM 400, remove the Aggrewell TM medium, refill 1ml with 107 The suspension of iPSCs in cell / ml was centrifuged and cultured in an incubator for 24 hours. The next day, in a biosafety cabinet, the EBs were aspirated out and stained with Aggrewell in a low-adhesion six-well plate. TM medium (containing 0.3mM Ascorbic acid (ascorbic acid, referred to as AA, from the American company Sigma), 10ng / ml Recombinant Human TGF-beta 2 Protein (transf...

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Abstract

The invention relates to a method for constructing a 3D pseudo-epidermis based on organ chips and directed differentiation of induced pluripotent stem cells, comprising the following steps: placing a 3D spinning scaffold inoculated with fibroblasts on one side and keratinocytes on the other side in an inert environment. Into the organ chip after bacteria, two kinds of media, CnT-07 epidermal keratinocyte medium and DMEM (containing 10% volume ratio FBS, 1% volume ratio penicillin-streptomycin), were passed through to culture keratinocytes and fibroblasts respectively , to achieve co-culture and cell proliferation of keratinocytes and fibroblasts. In the construction method of the present invention, the required cells are obtained by using the stem cell differentiation technology, the cells are inoculated on the nanostructure, and the co-cultivation of keratinocytes and fibroblasts is realized in the organ chip, so that the repair of epidermal tissue has become a current research hotspot. and new methods for drug modeling.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a 3D pseudo-epidermis based on organ chips and directed differentiation of induced pluripotent stem cells. Background technique [0002] The skin is the largest organ of the human body, mainly responsible for the functions of protecting the body, sweating, feeling cold and heat, and pressure. Human skin is mainly composed of three layers: the epidermis, the dermis and the subcutaneous tissue. The keratinocytes and fibroblasts in the dermis can secrete a variety of macromolecular substances such as collagen, fibronectin, laminin and proteoglycans. In daily life, skin damage and skin diseases are inevitable. Therefore, a large number of durable skin substitutes are needed to repair the damaged epidermis. At the same time, the epidermal disease model is established to conduct drug administration research and determine the effect of drugs on epidermal diseases. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/0698C12N2502/094C12N2502/1323C12N2513/00C12N2533/30
Inventor 张玮莹肖婷昱吴轩曹一平
Owner JIANGHAN UNIVERSITY
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