Mouse primary hepatocyte perfusion type separating and in-vitro culturing method

Abstract

The invention discloses a mouse primary hepatocyte perfusion type separating and in-vitro culturing method. The perfusion type separating of mouse primary hepatocytes is conduced through an in-situ living body perfusion method, a high-yield single-cell suspension can be obtained, and therefore a basis is provided for in-vitro long-time culturing of the primary hepatocytes. An applied stimulating culture solution contains mouse colony stimulating factors (CSF), mouse IL-2, mouse IL-6 and mouse recombinant hepatocyte growth factors r-mHGF and other cell factors; the primary hepatocytes can be separated through stimulation according to the reasonable ratio for in-vitro proliferation, and the number can reach 2.25 times the number of initially-added primary hepatocytes 72 hours later. The cellproliferation activity can be kept for at least 1-2 weeks under the in-vitro conditions, the problem that the primary hepatocytes cannot be cultured for a long time in vitro is solved, and thereforethe method can be used for infection of retroviruses, provides a basis for over-expression target genes or knockout target genes in in-vitro primary cells, and greatly expands the application field ofthe primary hepatocytes.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for perfusion separation and in vitro culture of primary mouse hepatocytes. Background technique [0002] The study of liver-related diseases and their metabolism requires obtaining normal primary hepatocytes from liver tissue and culturing them in vitro. The use of primary hepatocytes for in vitro experiments has its own significant advantages compared to other methods of in vivo and in vitro experiments, because primary cultured hepatocytes are not affected by the complex effects of the neuroendocrine system in vivo, and can also maintain Specific functions of hepatocytes. [0003] The isolation of hepatocytes was initially performed by the isolated perfusion method. By 1975, Seglen successfully isolated rat hepatocytes with high viability using the in situ two-step collagenase perfusion method. Compared with the previous isolated liver perfusion, in...

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Problems solved by technology

However, because the hepatocytes are bound together by tight junctions, they cannot be made into a single-cell suspension by conventional methods such as cutting, grinding, and filtering, and it is difficult to form high-yield single-cell suspensions by enzymatically hydrolyzing liver t

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