Method for functionally detecting antibody-mediated organ rejection, application of method and kit
A technology for detecting antibodies and functionalities, applied in the biological field, can solve the problems such as the inability of organs to perform their expected functions and increase the economic burden of patients, and achieve the effect of reliable diagnosis basis, high sensitivity and accuracy
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Embodiment 1
[0072] Example 1 Separation of vascular endothelial cells
[0073] Solid tissues obtained aseptically through surgical procedures are placed in rich media and transferred to the processing room within 3-5 hours. Depending on the nature of the entity organization, different solutions are adopted.
[0074] (1) For incomplete tissues such as blood vessel fragments, fat, muscle, skin, some organs, and umbilical cord, cells will be collected by direct digestion. The specific steps are: cut the tissue into pieces of 1 cubic millimeter, wash and remove all the blood, place the tissue in a culture medium containing 1% collagenase by volume, and incubate at 37°C for 0.5-1 hour , mechanically loosen the digested tissue and separate single cells and residual tissue with gauze. Collagenase was removed using centrifugation.
[0075] (2) For intact organs with vascular connections such as liver, lung, kidney, etc., collect vascular endothelial cells by organ perfusion. According to the ...
Embodiment 2
[0076] Example 2 Culture of vascular endothelial cells
[0077] The single cell suspension obtained in Example 1 was placed in a mixture containing 20% fetal bovine serum by volume, 15 mg / ml endothelial cell growth stimulating supplement (ECGS), 50 units / ml heparin, 100 units / ml penicillin-100 units / ml M1 streptomycin culture medium, inoculated in a culture bottle coated with 1% gelatin, placed at 35°C and 4% CO by volume. 2 Culture in a saturated humidity incubator, and replace the culture medium after 24 hours.
Embodiment 3
[0078] Example 3 Cryopreservation of vascular endothelial cells
[0079] When the growth of the cells reached to cover 90% of the surface area of the culture flask, the cells were detached from the surface of the culture flask with a concentration of 0.05% trypsin-ethylenediaminetetraacetic acid disodium salt (EDTA) digestion solution. After centrifuging to remove residual enzymes, suspend the cells in a culture medium containing 5% DMSO by volume, gradually reduce the temperature of the above liquid to -80°C at a rate of 1-2°C / min, and then transfer to liquid nitrogen Store in can.
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