Organoid is a cell organization grown in a three-dimensional (3D) culture system which represents all characteristics of its origin. However, this organ-like structure requires supporting matrix to maintain its characteristics and functions. Matrigel, derived from mouse sarcoma, has often been used as the supporting matrix for organoids, but the result may not be desirable for clinical applications because of the unidentified components from the mouse sarcoma. On the other hand, natural characteristics of collagen emphasize toxic-free friendly niche to both organoid and normal tissue. Hence, this study attempts to develop a new, collagen-based matrix that may substitute Matrigel in organoid culture. Collagen-based matrix was made, using type 1 collagen, Ham’s F12 nutrient mixture, and bicarbonate. Then, characteristics of mouse colon organoids were analyzed by morphology and quantitative messenger RNA (mRNA) expression, revealing that the mouse colon organoids grown in the collagen-based matrix and in Matrigel had quite similar morphology, specific markers, and proliferative rates. Mouse small intestine–derived organoids, stomach-derived organoids, and human colon–derived organoids were also cultured, all of which were successfully grown in the collagen-based matrix and had similar properties compared to those cultured in Matrigel. Furthermore, possibility of organoid transplantation was observed. When mouse colon organoids were transplanted with collagen matrix into the EDTA-colitis mouse model, colon organoids were successfully engrafted in damaged tissue. For that reason, the use of collagen-based matrix in organoid culture will render organoid cultivation less expensive and clinically applicable.

We developed a novel dentin-pulp-like organoid. It has both stem-cell and odontoblast characteristics using a mesenchymal cell lineage of human dental-pulp stem cells (hDPSCs). The mixture of hDPSCs and Matrigel was transferred into the maintenance medium (MM) and divided into four different groups according to how long they were maintained in the odontogenic differentiation medium (ODM). All organoids were harvested at 21 days and analyzed to find the optimal differentiation condition. To assess the re-fabrication of dentin-pulp-like organoid, after dissociation of the organoids, it was successfully regenerated. Additionally, its biological activity was confirmed by analyzing changes of relevant gene expression and performing a histology analysis after adding Biodentine® into the ODM. The organoid was cultured for 11 days in the ODM (ODM 11) had the most features of both stem cells and differentiated cells (odontoblasts) as confirmed by relevant gene expression and histology analyses. Micro-computed tomography and an electron microscope also showed mineralization and odontoblastic differentiation. Finally, ODM 11 demonstrated a biologically active response to Biodentine® treatment. In conclusion, for the first time, we report the fabrication of a dentin-pulp-like organoid using mesenchymal stem cells. This organoid has potential as a future therapeutic strategy for tooth regeneration.

Background Tear deficiency due to lacrimal gland (LG) dysfunction is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue; however, the number of stem cells in the LG tissue is low, and 2D long-term cultivation reduces the differentiation capacity of stem cells. Nevertheless, 3D LG organoids could be an alternative for a DED therapy because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. Here, we report the development of LG organoids and their application as cell therapeutics. Methods Digested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, specific marker expression, and histological characteristics were analyzed to authenticate the formation of LG organoids. The secretory function of LG organoids was confirmed  through calcium influx or proteomics analysis after pilocarpine treatment. To explore the curability of the developed organoids, mouse-derived LG organoids were fabricated and transplanted into the lacrimal tissue of a mouse model of DED. Results The histological features and specific marker expression of LG organoids were similar to those of normal LG tissue. In the pilocarpine-treated LG organoid, levels of internal Ca2+ ions and β-hexosaminidase, a lysosomal protein in tear fluid, were increased. In addition, the secreted proteins from pilocarpine-treated lacrimal organoids were identified through proteomics. More than 70% of the identified proteins were proven to exosome through gene ontology analysis. These results indicate that our developed organoid was pilocarpine reactive, demonstrating the function of LG. Additionally, we developed LG organoids from patients with Sjogren’s syndrome patients (SS) and confirmed that their histological features were similar to those of SS-derived LG tissue. Finally, we confirmed that the mouse LG organoids were well engrafted in the lacrimal tissue two weeks after transplantation. Conclusion This study demonstrates that the established LG organoids resemble the characteristics of normal LG tissue and may be used as a therapy for patients with DED.

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Colon organoids (colonoids) are known to be similar to colon tissue in structure and function, which makes them useful in the treatment of intestinal de‐epithelialized disease. Matrigel, which is used as a transplantation scaffold for colonoids, cannot be used in clinical applications because of its undefined composition and tumorigenicity. This study identifies clinically available scaffolds that are effective for colonoid transplantation in damaged intestinal mucosa. The colon crypt was isolated and cultured from C57BL/6‐Tg[CAG enhanced green fluorescent protein (EGFP)1310sb/LeySopJ mice into EGFP + colonoids and subsequently transplanted into the EDTA colitis mouse model using gelatin, collagen, or fibrin glue scaffolds. To identify scaffolds suitable for colonoid engraftment in injured colon mucosa, the success rates of transplantation and secondary EGFP colonoid formation were measured, and the scaffolds' mediated toxicity in vitro and in vivo was observed in recipient mice. When colonoids were transplanted with gelatin, collagen, and fibrin glue into the EDTA colitis mouse model, all groups were found to be successfully engrafted. Fibrin glue, especially, showed significant increase in the engrafted area compared with Matrigel after 4 wk. The scaffolds used in the study did not induce colonic toxicity after transplantation into the recipients' colons and were thus deemed safe when locally administrated. This study suggests new methods for and provides evidence of the safety and utility of the clinical application of colonoid‐based therapeutics. Furthermore, the methods introduced in this study will be helpful in developing cell treatment using the esophagus or a stomach organoid for various digestive‐system diseases.—Jee, J., Jeong, S. Y., Kim, H. K., Choi, S. Y., Jeong, S., Lee, J., Ko, J. S., Kim, M. S., Kwon, M.‐S., Yoo, J. In vivo evaluation of scaffolds compatible for colonoid engraftments onto injured mouse colon epithelium. FASEB J. 33, 10116–10125 (2019). http://www.fasebj.org