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Culture method of building kidney-like organ by utilizing human multipotent stem cells

A technology for human pluripotent stem cells and cell culture, which is applied in the field of using human induced pluripotent stem cells to build kidney-like organs, and can solve the problems of building kidney-like organs with human pluripotent stem cells.

Inactive Publication Date: 2017-05-31
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no technical report on the use of human pluripotent stem cells to construct kidney-like organs in vitro

Method used

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  • Culture method of building kidney-like organ by utilizing human multipotent stem cells
  • Culture method of building kidney-like organ by utilizing human multipotent stem cells
  • Culture method of building kidney-like organ by utilizing human multipotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Construct kidney organoids according to the following operations (all methods in the examples are illustrated by taking one well of a 6-well plate as an example):

[0046] 1) Matrigel is used as the extracellular matrix and spread on the surface of the cell culture dish

[0047] Take 1ml of Matrigel (Basement Membrane Matrix, purchased from Beijing Saibei Biotechnology Co., Ltd.) with a pipette gun, transfer it into a cell culture dish, shake it well, make the Matrigel evenly cover the bottom of the culture dish, and then cover the Matrigel Place the cell culture dish at 37°C for 4 hours, and the matrigel should keep completely covering the bottom of the dish during the placement.

[0048] 2) Remove Matrigel, add feeder-free medium to the cell culture dish, inoculate human pluripotent stem cells, and carry out human Culture and passage of pluripotent stem cells

[0049] Specifically, proceed as follows:

[0050] Remove the matrigel in the new culture dish used i...

Embodiment 2

[0062] Construct kidney organoids according to the following operations (all methods in the examples are illustrated by taking one well of a 6-well plate as an example):

[0063] 1) Matrigel is used as the extracellular matrix and spread on the surface of the cell culture dish

[0064] Take 1ml of matrigel hESC qualified Matrigel (In VitroTechnologies, #FAL354277) with a pipette gun, move it into the cell culture dish, shake and shake well, so that the matrigel is evenly distributed on the bottom of the culture dish, and then place the matrigel-covered cell culture dish Place overnight at 2-8°C, and the matrigel should keep completely covering the bottom of the dish during the placement.

[0065] 2) Remove Matrigel, add feeder-free medium to the cell culture dish, inoculate human pluripotent stem cells, and carry out human Culture and passage of pluripotent stem cells

[0066] Specifically, proceed as follows:

[0067] Remove the matrigel in the new culture dish used in ...

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Abstract

The invention provides a culture method of building a kidney-like organ by utilizing human multipotent stem cells. The culture method comprises the following steps: 1) taking matrix gelatin as an extracellular matrix, and laying the matrix gelatin on the surface of a cell culture plate; 2) removing the matrix gelatin, adding a feeder layer free medium in the cell culture plate, inoculating the human multipotent stem cells, and carrying out passage culture on the human multipotent stem cell; 3) when the cell confluence reaches 40-50 percent, removing the feeder layer free medium, adding a basal medium containing cell factors, and carrying out culture and differentiation on the human multipotent stem cells; and 4) collecting cells, putting the cells in a Transwell culture device, and preparing the kidney-like organ. The invention also provides the kidney-like organ prepared according to the method. The kidney-like organ is formed by endothelium and renal interstitium surrounding an intact segmented kidney units, and transcription of the kidney-like organ is similar to that of a human embryonic kidney.

Description

technical field [0001] The invention relates to a method for culturing kidney-like organoids, in particular to a method for constructing kidney-like organoids using human induced pluripotent stem cells (hiPSCs). Background technique [0002] Stem cells are cells capable of differentiating into various cell types. Human induced pluripotent stem cells (hiPSCs) can differentiate into a variety of cell types, and now they can directly differentiate into organoids such as brain, heart, intestine, liver, lung, and kidney. Despite remarkable achievements in the construction of tissue-specific units and organoids, preserving such cell aggregates in vitro remains a formidable challenge. Cell culture requires oxygen and nutrients, but 3D organoids constructed in vitro lack vasculature, so their lifespan, size, and maturity are often limited. [0003] The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. In order to fulfill functions such as...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2501/119C12N2501/91C12N2506/45
Inventor 肖枫林李清刚陈香美
Owner GENERAL HOSPITAL OF PLA