NOVEL CULTURE AND ORGANOID TECHNOLOGIES TO STUDY MAMMALIAN KIDNEY DEVELOPMENT, ACTA UNIVERSITATIS OULUENSIS D Medica 1454
|Kustantaja:||Oulun yliopisto|| |
|Painos:||Osajulkaisuväitöskirjan yhteenveto-osa|| |
|Sijainti:||Print Tietotalo|| |
|Tekijät:||SAARELA ULLA|| |
Kidney diseases affect an increasing number of people worldwide, and there is a growing demand
to develop new treatments and increase the number of transplantable organs. New treatments can
be designed when new knowledge is gained by studying the details of kidney development. The
ex vivo culture techniques have been used for over a century to study the development of kidneys,
but they are not optimal for long-term imaging and following the nephrogenesis process over time.
Kidney organoids, which are cellular aggregates resembling the in vivo kidney, together with
intact embryonic kidneys, present a platform for these studies. However, there are limitations
when working with primary embryonic kidney cells. Primary embryonic metanephric
mesenchymal cells are usually low in number and lose the ability to undergo nephrogenesis
rapidly. New ways to culture, biobank, and transfect cells can offer ways for functional testing of
the effects of different genes on the nephrogenesis.
This study presents new tools for studying nephrogenesis. Time-lapse imaging of organ
development may be enhanced by using a Fixed Z-direction (FiZD) culture system where the
kidney explant is grown in a restricted 70μm space. The technique enables the segmentation of the
individual cells in a two-dimensional image and a dynamic analysis of the time-lapse data.
This study also presents a technique of dissociation and reaggregation of the uninduced kidney
metanephric mesenchyme (MM). With this novel method of culturing the dissociated MM cells in
a growth factor medium for 24 hours, the cells can keep their competence for nephrogenesis. This
technique allows the genetic manipulation of the MM cells before the induction to form nephrons,
allowing functional testing of genes in the metanephric mesenchyme.
This study further presents different techniques for gene editing of MM cells and introduces
biobanking of primary kidney cells. It is shown here that the MM and ureteric bud (UB) cells have
the capability to remember their fates and build nephron-like structures or continue branching after
the cryopreservation in the liquid nitrogen. The methods introduced here provide new ways to
create kidney organoids, manipulate their genome, and biobank the primary embryonic kidney
cells. The developed FiZD culture system enhances the imaging of kidney development compared
to the previously used culture methods. Using this method, the morphogenesis of the developing
kidney can be followed more precisely, even in a single cell level. This culture method may also
be used to culturing other organs, such as ovary, and may help provide insights into the
development of other tissues as well.