A method for digestion and dissociation of primary tissues of retinal pigment epithelial cells

A technology for retinal pigment epithelial cells, applied in the field of digestion and dissociation of primary tissues of retinal pigment epithelial cells, which can solve the problems of loss of human retinal pigment epithelial cell function expression, variable separation efficiency, poor culture effect, etc. problems, achieve the effect of reducing the influence of non-RPE cells, increasing the purity, and shortening the digestion time

Active Publication Date: 2022-05-31
EYECURE THERAPEUTICS INC JIANGSU
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The primary tissue separation technology of retinal pigment epithelial cells currently on the market mainly relies on enzyme digestion, which generally takes a long time, and the number of cells removed is relatively small, the separation efficiency varies between batches, and the culture effect is not good. The yield of cells is different, so the loss of specific function expression of human retinal pigment epithelial cells is relatively large

Method used

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  • A method for digestion and dissociation of primary tissues of retinal pigment epithelial cells
  • A method for digestion and dissociation of primary tissues of retinal pigment epithelial cells
  • A method for digestion and dissociation of primary tissues of retinal pigment epithelial cells

Examples

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Embodiment 1

[0044] Embodiment 1: Human retinal pigment epithelial cell (RPE) digestion and dissociation method of the present invention

[0045] Configure RPE medium according to the recipe in Table 1.

[0046] Table 1: RPE medium used in the present invention

[0047] components content α-modified minimal essential medium (MEM, αmodification) 500mL N1 additive (N1 supplement) 5mL Glutamine 5mL Non-essential amino acids 5mL Taurine 150mg Hydrocortisone 15μg Triiodothyronine 0.010μg Human Platelet Lysate (HPL) 50mL

[0048] Follow the steps below to digest and dissociate primary human retinal pigment epithelial tissue:

[0049] (1) Uncover the sealing film of the 15ml centrifuge tube containing the eye tissue in a biological safety cabinet, suck out the eye tissue with a 1ml pipette, and put it into two tubes of DPBS (CTS) in the prepared twelve-well plate. Wash in the well, 3-5s each time.

[0050] (2) Put the eye ti...

Embodiment 2

[0060] Example 2: Prior art, traditional human retinal pigment epithelial (RPE) digestion and dissociation method

[0061] Embodiment 2 performs synchronous operation with embodiment 1.

[0062] The same human retinal pigment epithelial cell culture medium was configured according to the recipe in Table 1 in Example 1.

[0063] Referring to the prior art, digest and dissociate human retinal pigment epithelial cells according to the following steps:

[0064] (1) Uncover the sealing film of the 15ml centrifuge tube containing the eye tissue in a biological safety cabinet, suck out the eye tissue with a 1ml pipette, and put it into two tubes of DPBS (CTS) in the prepared twelve-well plate. Wash in the well, 3-5s each time.

[0065] (2) Put the eye tissue into a 100mm petri dish with about 10ml of DPBS (CTS), under a stereomicroscope, trim off excess tissue on the surface, and move the trimmed eye tissue into another culture dish filled with about 10ml of DPBS (CTS). Cut off th...

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Abstract

The invention discloses a method for digestion and dissociation of primary tissues of retinal pigment epithelial cells, which comprises the following steps: turning the retinal layer in the eyecup to the outside and then putting it into digestive enzymes, so that the digestive enzymes can affect the retinal pigment epithelium The influence of the cells is reduced to the minimum, and more retinal pigment epithelial cells are obtained while ensuring the purity of the retinal pigment epithelial cells. This method has wide application prospects.

Description

technical field [0001] The invention generally relates to the technical field of cell engineering, in particular to a method for digesting and dissociating primary tissue of retinal pigment epithelial cells. Background technique [0002] RPE (human retinal pigment epithelium) is a continuous monolayer of cells located outside the retina, named for its sieve-like morphology and abundant melanin in the cytoplasm, they play an important role in the maintenance and self-renewal of retinal cells , such as phagocytosis and digestion of the outer segment (OS) of exfoliated photoreceptor cells, promoting the regeneration of 11-cis retinal, an important mediator in the visual cycle, regulating the immune response in the eye, participating in the formation of the retina-vascular barrier, etc. The abnormality of this physiological function of RPE cells is considered to be closely related to the occurrence of related eye diseases. For example, abnormal RPE cell barrier function can easi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079
Inventor 方攀峰谢磊范国平王云娟
Owner EYECURE THERAPEUTICS INC JIANGSU
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