Long-term storage of human fetal haematopoietic progenitor cells and their subsequent reconstitution. Implications for in utero transplantation.
Haematopoietic progenitor cells were isolated from human fetal liver, obtained between 6 and 15 weeks gestation. After preparation of a single cell suspension, the cells were stored using a stepwise freezing protocol; taking the cells from room temperature through -70 degrees C to liquid nitrogen. Viability (trypan blue exclusion), morphology (Leishman stain), identification of cell type (flow cytometry) and growth characteristics in semi-solid culture medium were assessed using the fresh cell suspension. We were able to confirm that the predominant cells in human fetal liver up to about 15 weeks gestation are those of the erythroid lineage. It was established that viability in excess of 75% was required to ensure adequate growth in culture after frozen storage and it was deemed important to ensure morphological integrity of the cell preparations. The colonies formed in culture were observed to be producing haemoglobin between 7 and 9 days after initial seeding. We have determined that cells can be stored in liquid nitrogen for up to 2 years without loss of (1) viability, (2) morphological features and (3) ability to form colonies and produce haemoglobin in culture. These findings offer encouragement for the implementation of a cell bank to support an in utero transplantation programme.
Jones
D R
DR
Department of Immunology, Queens Medical Centre, Nottingham, UK.
Anderson
E M
EM
Evans
A A
AA
Liu
D T
DT
D002469:Cell Separation; D002478:Cells, Cultured; D015925:Cryopreservation; D004920:Erythropoiesis; D005260:Female; D005315:Fetal Diseases / Q000628:therapy*; D016332:Fetal Tissue Transplantation; D018380:Hematopoietic Stem Cell Transplantation; D006453:Hemoglobinopathies / Q000628:therapy*; D006801:Humans; D011247:Pregnancy