Biomedical Science and Engineering 2019; volume 3(s2):102

Organoid culture systems: Products supporting one of the Materials and Methods Correspondence: Salvatore Simmini, STEM- CELL Technologies UK Ltd., Cambridge, biggest revolutions in biological IntestiCult™ OGM (Mouse) UK. research Upper intestines of C57BL6/J mice E-mail: [email protected] were dissected and washed several times in PBS. The intestinal fragments were then Key words: ; IntestiCult; intestine; Ryan Conder,1 Leon H. Chew,1 incubated for 20 minutes with Gentle Cell ; STEMdiff. Fisal Elstone,1 Marianne Lankhorst,1 Dissociation Reagent (GCDR) at room tem- Conference presentation: this paper was pre- 1 3 Adam Añonuevo, Salvatore Simmini, perature (RT) to separate the crypts and villi sented at the Second Centro 3R Annual Wing Chang,3 Allen C. Eaves,1,2 from the intestinal basal surface. The crypts Meeting - 3Rs in Italian Universities, 2019, Terry E. Thomas,1 Sharon A. Louis1 were then isolated from villi through cen- June 20-21, University of Genoa, Italy. 1 trifugation, counted and re-suspended in a STEMCELL Technologies Inc., 50:50 mixture of Corning® Matrigel® dome Received for publication: 28 October 2019. 2 Vancouver, Canada; Terry Fox and IntestiCult™ OGM at 6,000 crypts/mL. Accepted for publication: 6 November 2019. Laboratory, BC Cancer Agency, A 50 µL droplet of the suspension was gen- This work is licensed under a Creative 3 Vancouver, Canada; STEMCELL tly placed into the center of pre-warmed 12- Commons Attribution NonCommercial 4.0 Technologies UK Ltd., Cambridge, UK well culture plate wells, creating a dome License (CC BY-NC 4.0). containing ~300 crypts/well. The domes were solidified at 37°C for 5 min and the ©Copyright: the Author(s), 2019 wells were then flooded with 750 µL of Licensee PAGEPress, Italy IntestiCult™ OGM. Crypts were cultured at Biomedical Science and Engineering 2019; 3(s2):102 doi:10.4081/bse.2019.102 Abstract 37°C for 4-7 days with 3 medium changes Organoids are -derived struc- per week. After 7 days, organoids were pas- Stage 3). The expanded organoids were tures that are generated in three-dimension- saged by treating cultures with GCDR for thenonly cultured in Maturation Medium, with al tissue culture. They are unique since they 15 min at RT followed by mechanical dis- agitation, for extended periods of time (days exhibit a high degree of self-organization ruption into smaller aggregates. The result- ant suspension was mixed with 10 - 40+, Stage 4). Morphological analysis and differentiation, and thus recapitulate IntestiCult™ OGM at a 1:6 ratio and then of organoids was performed on days 5, 7, 10 many of the features of the tissues from re-plated as above to establish secondaryuse and 40, which are the endpoints of Stages 1 which they were derived. Because of this, cultures. This protocol was repeated to gen- - 4 respectively. Organoids at day 40 were organoids are now firmly established as an erate long-term cultures. analyzed by RT-qPCR or cryosectioned and essential tool in medical research, and have processed for immunofluorescence (>3 the potential to drastically reduce the num- IntestiCult™ OGM (Human) organoids per analysis; 2 human embryonic ber of animals required for experimentation. Human intestinal crypts were isolated stem cell (hESC) lines, n = 2 and 2 induced by incubating human small intestine and pluripotent stem cell (iPSC) lines, n = 2). colon tissue samples with GCDR for 30 minutes at 4°C with gentle agitation. The Introduction liberated crypts were counted and plated at ® ® Results Organoids are stem cell-derived struc- 1,000 crypts per Corning Matrigel dome, tures that are generated in three-dimension- flooded with 750 µL IntestiCult™ OGM IntestiCult™ OGM (Mouse) al tissue culture. Organoids are unique since (Human) and cultured at 37°C with 3 medi- The efficiency of mouse intestinal they exhibit an advanced degree of self- um changes per week. The mature organoids generation in IntestiCult™ OGM organization and differentiation, and thus organoids were expanded by harvesting, (Mouse) was 64 ± 8% (mean ± SD; n=6) by dissociated by manual agitation and 10 min recapitulate many of the features of the tis- day 5, yielding ~190 organoids per well. GCDR incubation and re-plated at a 1:4 sues from which they are derived.Non-commercial Because Organoids generally consist of an inner split ratio every 10-12 days over 15 pas- of their similarity to their in vivo counter- lumen surrounded by a complex arrange- sages. parts, organoids are emerging as the pre- ment of multiple crypt-like buds. formation efficiency increased to 86±4% ferred model system for studying tissue STEMdiff™ Cerebral Organoid Kit (n=4) after the first passage and remained development, homoeostasis, and diseased This kit contains 2 basal media and 5 consistently >85% over at least 12 passages states in vitro. Organoid technology is wide- supplements, which are combined to pre- (90±3%, n=3). Physiological relevance was ly recognized as one of the greatest techno- pare four separate complete media corre- addressed by demonstrating the presence of logical breakthroughs in basic biological sponding to the 4 stages of cerebral intestinal stem cells (Lgr5), polarized ente- research in the last decade. For these rea- organoid formation. Human pluripotent rocytes (Villin), goblet cells (Muc2), sons, this technology holds tremendous stem cells (hPSCs) maintained in enteroendocrine cells (Chg A), and Paneth potential to complement 2D-culture meth- mTeSR1™ were dissociated into single cell cells (Lysozyme) by immunocytochemistry ods and highly reduce the use of animal suspensions, and cultured in Embryoid and qPCR analysis of organoids at each pas- models in research. STEMCELL Body (EB) Formation Medium (days 1 - 5, sage. Technologies is committed to developing Stage 1). The resulting EBs were then trans- culture media and providing tools that effi- ferred to Induction Medium (days 6 - 7, IntestiCult™ OGM (Human) ciently support the generation and mainte- Stage 2); next, they were expanded by Spherical human intestinal organoid nance of multiple organoid culture systems. embedding in Corning® Matrigel® and cul- structures could be identified within 2 days tured in Expansion Medium (days 7 - 10, of initial seeding. Organoids analyzed by

[Biomedical Science and Engineering 2019; 3(s2):102] [page 40] Article immunohistochemical and qRT-PCR analy- iPSCs, n = 2) for EB generation (100% suc- ses for intestinal stem (LGR5 and AXIN2), cess, n = 128/128), expansion (>95% exhib- Conclusions Paneth (LYZ), enteroendocrine (CHGA), ited extensive folding of neuroepithelia, n = STEMCELL Technologies supports the goblet (MUC2) and enterocyte (VIL1) cell 104/107) and maturation (>60% of organoid culture system revolution in bio- marker expression, revealed that organoids organoids were >1 mm in diameter with logical research by developing products like were comprised of both stem and differenti- dense cores, n = 62/94). In vivo, the human IntestiCult™ OGM (Mouse and Human) ated cell types, demonstrating cultured cortex consists of progenitor and neuronal and STEMdiff™ Cerebral Organoid Kit human organoids closely resembled human populations that organize into distinct lay- that enable generation of organoids in a intestine structure. Organoids expanded for ers. The mature organoids exhibited a simi- highly reproducible manner, thus reducing >1 year in culture with an average passag- lar architecture with neural progenitors experimental variability and increasing ing ratio of 1:6 every 8 to 12 days. (SOX2+, PAX6+) localized in apical regions accuracy of results for researchers to surrounding a central ventricle. Adjacent to advance their scientific discoveries. STEMdiff™ Cerebral Organoid Kit the apical progenitors, neuronal progenitors The efficiency of the STEMdiff™ (TBR2+, Ki-67+) were found abutting neu- Cerebral Organoid Kit was tested across rons (CTIP2+, MAP2+, TBR1+), resembling multiple cell lines (2 hESCs, n = 2 and 2 the intermediate zone and cortical plate regions.

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