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Method for rapidly constructing multilayer cells

A technology of layered cells and seed cells, which is applied in the field of tissue engineering, can solve the problems of excessive cell differentiation, aging, and restrictions on clinical application, and achieve the effects of good product reproducibility, simple and flexible process, and industrialization

Inactive Publication Date: 2015-04-15
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

② Imperfect biological and physical functions: the current in vitro bioreactors cannot fully simulate the real microenvironment faced by cells growing under in vivo conditions. Conditions vary widely
Therefore, a longer construction time usually leads to excessive cell differentiation and aging in the constructed cell layer, and the adhesion and proliferation functions of various cells will continue to decline. At the same time, various physical functions of the cell layer (biological functions of the cell layer Mechanical strength, electrophysiological function, tissue polarity, etc.) will also decrease accordingly
The various functional deficiencies of these constructed tissues also seriously restrict the final clinical application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Rapid preparation of tissue-engineered lamellar cornea using acellular keratoma gel and acellular keratoma scaffold.

[0028] (a) Preparation of seeded cells.

[0029] Four pieces of 2mm tissue blocks from the superior temporal quadrant of the limbus were taken, routinely cultured in the primary culture and subcultured to the P3 generation, and the obtained 3×10 6 Corneal epithelial cells were placed in a centrifuge tube, the culture medium was removed by centrifugation, and the corneal epithelial cells were used as seed cells for future use.

[0030] (b) Prepare the acellular corneal stroma gel, and control the concentration and temperature to make it liquid.

[0031]The decellularized corneal stroma sample prepared by the conventional method was weighed and added with 1 mg / mL pepsin (pepsin) + 0.1 mol / L HCL (DDW, pH=1) at a ratio of 1:5 v / v, and dissolved in a water bath at 50°C. After a large amount of basic amino acids are completely dissolved, the pH value of the...

Embodiment 2

[0040] Rapid preparation of tissue-engineered skin cell sheets using acellular skin matrix gel.

[0041] (a) Preparation of seeded cells.

[0042] Four tissue pieces with a diameter of 2mm obtained from living organisms were taken, and they were conventionally cultured in the primary culture respectively, and subcultured and amplified to the P5 generation to obtain 4×10 7 human epithelial cells and 1×10 7 For individual fibroblasts, mix human epithelial cells and human fibroblasts at a ratio of 4:1, centrifuge to remove the culture medium, and use it as seed cells for later use.

[0043] (b) Prepare the acellular skin matrix gel, and control the concentration and temperature to make it liquid.

[0044] Acellular skin matrix samples (prepared according to conventional methods) were weighed and added 1 mg / mL pepsin (pepsin) + 0.1 mol / L HCL (DDW, pH = 1) at a ratio of 1:5 v / v, dissolved in a water bath at 50°C, and used 1 mol / L NaOH (DDW, pH=14) solution to adjust the pH valu...

Embodiment 3

[0053] Rapid preparation of tissue-engineered cardiomyocyte sheets using decellularized cardiac matrix gel.

[0054] (a) Preparation of seeded cells.

[0055] Freshly isolated 1-day-old suckling mouse ventricular muscle tissue, the obtained 2×10 6 A cardiomyocyte was placed in a centrifuge tube, and the culture medium was removed by centrifugation, and set aside.

[0056] (b) Prepare the decellularized cardiac matrix gel, and control the concentration and temperature to make it liquid.

[0057] Decellularized heart matrix samples (prepared according to conventional methods) were weighed and added 1mg / mL pepsin (pepsin) + 0.1mol / L HCL (DDW, pH=1) at 1:5v / v, dissolved in a water bath at 50°C, and used 1mol / L NaOH (DDW, pH=14) solution to adjust the pH value of the sample to 7.4, add 10× cardiomyocyte culture medium at a volume ratio of 9:1, centrifuge at 12000 rpm for 10 min, and take the supernatant to filter and sterilize. After quantitative protein detection, adjust the c...

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Abstract

The invention discloses a method for rapidly constructing multilayer cells. The method comprises the steps of mixing and suspending seed cells and liquid extracellular matrix gel, putting the liquid suspension on a construction carrier to ensure that the liquid suspension on the carrier is solidified into solid gel and a multilayer cell structure is directly formed, and adding a culture solution to culture the multilayer cells till enough strength. The method disclosed by the invention is capable of rapidly preparing various tissues and organs which contain the multilayer cells and meet the requirements of proliferation activity and mechanical strength, is a breakthrough of tissue engineering construction technology and is reliable in principle, simple and flexible in process, good in product reproducibility and very easy in industrialization at the same time.

Description

Technical field: [0001] The invention belongs to the field of tissue engineering, and in particular relates to a method for quickly constructing stratified cells. Background technique: [0002] The core of tissue engineering is to establish a three-dimensional complex of cells and biomaterials to reconstruct the shape, structure and function of diseased or damaged tissues and achieve permanent replacement. For complex tissues and organs with multicellular structures, the basis of cell structure determines the functions of tissues and organs. How to prepare a viable multilayered cell structure plays an important role in the development of this field and clinical practical application. [0003] The conventional multilayer cell construction method is: planting cell layer-by-layer growth method. Firstly, a single layer of cells is planted. The underlying cells can adhere by means of electrostatic adsorption or binding with ligands of the extracellular matrix. After sufficient ti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071A61L27/28
Inventor 王智崇武征周强
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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