Your browser doesn't support javascript.
loading
Preparing single cell suspension of rat diaphragm tissues: Mechanical grinding versus mechanical-enzymatic digestion / 中国组织工程研究
Chinese Journal of Tissue Engineering Research ; (53): 1213-1217, 2020.
Article in Chinese | WPRIM | ID: wpr-847968
ABSTRACT

BACKGROUND:

Flow cytometry is currently the advanced cell analysis technique with single cell suspension as a research basis, but there is no report on the preparation method of single cell suspension of diaphragm tissue.

OBJECTIVE:

To explore the feasibility of preparing single cell suspension of rat diaphragm tissue by mechanical grinding method and mechanical-enzymatic digestion method and to compare cell number and viability of the cells obtained using different methods.

METHODS:

The fresh diaphragm tissues of Sprague-Dawley rats were harvested. Based on the mechanical method, trypsin, collagenase I, collagenase II, collagenase IV and their different combinations were used to digest and prepare the single cell suspension of diaphragm tissues. Cell morphology was observed; cell number and viability were determined by trypan blue staining. The living cells, inactivated cells, and cell aggregates were counted, and cell survival rate and concentration of the single cell suspension were calculated. Statistical analyses were performed thereafter. RESULTS AND

CONCLUSION:

(1) The single cell suspension with better cell dispersion, more complete morphology, clearer boundary, fewer impurities and cell debris, and cleaner background were obtained by mechanical-enzymatic digestion compared with mechanical grinding method. (2) The single cell suspension prepared by simple mechanical grinding method has low number of living cells, high number of inactivated cells, low survival rate and many cell aggregates. (3) The number of living cells and concentration of single cell suspension obtained by same-volume addition of trypsin, collagenase I and collagenase IV mixed enzymes based on the mechanical grinding method were the highest, with (1.0-2.0)×106 cells per 0.1 g diaphragm tissue. There were significant differences between mechanical-enzymatic digestion and mechanical grinding method in terms of living cells, inactivated cells, cell aggregates, cell survival rate and suspension concentration (P < 0.05). Moreover, the single cell suspension prepared by the same-volume addition of trypsin and collagenase IV had higher suspension concentration, higher cell survival rate, and less inactivated cells and cell aggregates. To conclude, the single cell suspension of diaphragm tissues could be prepared successful by both mechanical and mechanical-enzyme digestion methods. Mechanical-enzyme digestion is superior to simple mechanical grinding method, with the best single cell suspension after same-volume addition of trypsin, collagenase I and collagenase IV. This is the preferred method for preparation of single cell suspension of the diaphragm tissue.

Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: Chinese Journal of Tissue Engineering Research Year: 2020 Type: Article

Similar

MEDLINE

...
LILACS

LIS

Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: Chinese Journal of Tissue Engineering Research Year: 2020 Type: Article