Evaluating Cell Migration and Colony Formation

The migration of native HPCs and ASCs was assessed using the scratch wound healing assay. The cells were seeded onto a 12-well plate and cultured until fully confluent, then a 10 μL pipette tip was used to create the scratch wound. The cells were then cultured for 48 h, and microphotographs of the wells were taken at 4 time points: 0 h, 6 h, 24 h and 48 h using an inverted Leica DMi1 microscope equipped with a MC170 camera (Leica Microsystems, KAWA.SKA Sp. z o. o., Zalesie Gorne, Poland). After 48 h, the cells were fixed with 4% PFA and stained with a pararosaline solution for the purpose of photographic documentation. The scratch closure was calculated based on the Leica software scale bar (Leica Application Suite- LAS EZ, version 3.4.0), using photographs from 4 wells. To assess the clonogenic potential of native ASCs and HPCs, a colony-forming unit assay was performed as described previously [102 (link),103 (link)]. Briefly, 250 cells were seeded onto a 6-well plate and cultured with medium changes every 3 days. After 8 days, the cells were fixed with a 4% PFA solution and stained with a 2% pararosaline solution (Sigma Aldrich/Merck, Poznan, Poland). The number of colonies was counted, and any cluster of 50 cells or more was regarded as a colony.