A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations.
Cellular reaggregation methods have been used to generate in vitro organotypic cultures as a means to elucidate the cellular and molecular requirements of organogenesis. However, reproducibility from experiment to experiment has remained problematic and furthermore, current protocols do not support reaggregation of many important tissues. Here, using the thymus as a model organ, we present a novel reaggregation method termed "compaction reaggregation" that offers improved kinetics of reaggregation and greatly improved efficiency. Using compaction reaggregation we have been able to reaggregate the aorta-gonad- mesonephros region, a tissue that previously proved refractory to commonly used reaggregation methods, enabling the study of hematopoietic stem cell emergence and expansion. Additionally, compaction reaggregation permits the juxtaposition of different cell layers within the aggregated structure thus providing the means to study inductive interactions between different cell populations in vitro.
Sheridan
Julie M
JM
MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, United Kingdom.
Taoudi
Samir
S
Medvinsky
Alexander
A
Blackburn
C Clare
CC
D000818:Animals; D002449:Cell Aggregation / Q000502:physiology*; D019070:Cell Lineage; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D009924:Organ Culture Techniques / Q000379:methods; D011247:Pregnancy; D013950:Thymus Gland / Q000166:cytology* / Q000196:embryology / Q000378:metabolism
D049452:Green Fluorescent Proteins; D007633:Keratins