|Title:||Cryopreservation of spherical tumor microtissues|
|Authors :||Rimann, Markus|
Kelm, Jens M.
|Conference details:||3D cell culture, Congress, Dechema, Zurich, 14–16 March 2012|
|Language :||Englisch / English|
|Subject (DDC) :||570: Biologie|
610: Medizin, Gesundheit
|Abstract:||Cryopreservation of mammalian cells and tissues is key for a sustainable supply of cells for basic research, drug discovery and regenerative medicine. However, the current paradigm shift towards more organotypic 3-dimensional cell culture formats leads to new challenges for robust cryopreservation protocols of 3D tissue constructs for bio-banking. Simple freezing protocols as routinely used today for single cell suspensions are not appropriate. Cryopreservation media composition and incubation times have to be adapted to altered diffusion resistance due to cell-cell contacts and extracellular matrix. The aim of this study was to establish a robust automation-compatible method to cryopreserve spherical tumor microtissues with respect to viability and functionality. Microtissues of three different cell lines characterized by different growth profiles (HCT-116, DU-145, SNB-19) were produced in hanging drops. They were grown for 4 days in the droplet cultures prior transferring them into non-adhesive 96-well plates. 5 different cryopreservation media with and without a controlled rate freezer were tested. Regular ATP- and microtissue diameter size measurements were performed to monitor viability and growth profile of the microtissues. HE-stained microtissue sections were prepared to assess tissue integrity after the freezing process. Moreover, drug testing was done to determine whether the freezing process altered drug sensitivity compared to non-frozen control tumor microtissues. Comparing the survival rate with and without controlled rate freezer demonstrated that microtissues could be frozen also in standard -80°C freezers. Microtissues showed similar ATP contents and growth profiles. Compared to non-frozen microtissues, they required, however, a lag phase of about 2 days prior entering their characteristic growth profile. Furthermore, the 5 different freezing media used did not show any significant differences in microtissues viability and growth profile over time. Dose response curves of common toxins confirmed the biological functionality of thawed microtissues. In conclusion, a straight forward industrial-compatible method was established to cryopreserve tumor microtissues without altering drug sensitivity.|
|Departement:||Life Sciences und Facility Management|
|Publication type:||Konferenz: Poster / Conference Poster|
|Appears in Collections:||Publikationen Life Sciences und Facility Management|
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