Vives and Batlle-Morera Stem Cell Research & Therapy (2020) 11:72 https://doi.org/10.1186/s13287-020-1586-1 COMMENTARY Open Access The challenge of developing human 3D organoids into medicines Joaquim Vives1,2,3* and Laura Batlle-Morera4* Abstract The capacity of organoids to generate complex 3D structures resembling organs is revolutionizing the fields of developmental and stem cell biology. We are currently establishing the foundations for translational applications of organoids such as drug screening, personalized medicine and launching the future of cell therapy using organoids. However, clinical translation of organoids into cell replacement therapies is halted due to (A) a few preclinical studies demonstrating their efficacy and (B) the lack of robust, reproducible, and scalable methods of production in compliance with current pharmaceutical standards. In this issue of Stem Cell Research & Therapy [ref], Dossena and collaborators present a validated bioprocess design for large-scale production of human pancreatic organoids from cadaveric tissue in accordance with current good manufacturing practice. The authors also propose a set of specifications of starting materials and critical quality attributes of final products that are of interest to other developments provided that this type of medicines are different than any other medicinal product due to their complex composition and living nature of the active ingredient. Although large-scale production of functional cells secreting insulin is still a challenge, the development of methods such as the one presented by Dossena and collaborators contributes to move toward clinical use of organoids in the treatment of type 1 diabetes and opens avenues for future clinical use of organoids in degenerative pathologies. Keywords: Good manufacturing practice, Organoid, Pancreas, Regenerative medicine, Tissue engineering, Regulatory and quality compliance Background restricted to drug testing [2, 3] and initial cell replacement Organoids are three-dimensional (3D) cell structures strategies [4, 5]. However, the regulatory framework does grown in vitro from stem cells, primarily isolated either not define specific guides or rules to such type of entities from biopsies or from pluripotent stem cells, that recap- and, for instance, drug testing using organoids in Europe itulate key features of both the development and perform- needs be done in accordance with a very generic “Guideline ance of native organs [1]. Cells forming organoids are able on the principles of regulatory acceptance of 3Rs (replace- to differentiate, undergo lineage commitment, and acquire ment, reduction, refinement) testing approaches” (EMA/ the specific tissue patterning to develop into several endo- CHMP/CVMP/JEG-3Rs/450091/2012) by the European derm, mesoderm, and ectoderm-derived tissues, mimick- Medicines Agency (EMA). Currently, the clinical use of ing somehow their in vivo counterparts. The resulting organoids would need to meet the requirements for cell structures are complex and provide a unique opportunity and gene-based therapies and produced the following good to model human organ development and disease in a sys- manufacturing practices (GMP) as any other pharma- tem remarkably similar to that occurring in vivo. ceutical drug [6]. Despite the extensive use of organoids in basic research, In any case, organoids hold a huge potential in regen- their translational biomedical application is currently erative medicine purposes if current limitations are over- come, namely, (A) clonal variation, developmental stage, * Correspondence: jvives@bst.cat; Laura.Batlle@crg.eu pluri-/multi-potency, and chromosomal stability of stem 1 Cell Therapy Service, Blood and Tissue Bank (BST), Barcelona, Catalonia, cells; (B) reproducibility, accuracy, and scalability of the Spain 4 Core Facilities Program, Centre for Genomic Regulation (CRG), Barcelona, methodologies proposed; (C) meaningful functional as- Catalonia, Spain sessment of resulting organoids; and (D) preclinical Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vives and Batlle-Morera Stem Cell Research & Therapy (2020) 11:72 Page 2 of 4 validation [7]. Only if these constraints are conveniently that they are consistently followed at each step in the addressed, organoid technology will be readily translated manufacturing process) [6]. In fact, protocols for the de- into novel approaches for the replacement of severely velopment of human-induced pluripotent stem cell damaged organs, such as the pancreas in patients with (iPSC)-derived retinal organoids have been successfully type 1 diabetes for instance. Indeed, the generation of adapted to GMP standards required for the generation human pancreas organoids (hPOs) in compliance with of transplantable photoreceptor cells for future clinical pharmaceutical standards is a challenge in view that cur- applications [11]. Similarly, Takebe and collaborators rents methods are laborious, time-consuming, expensive have further developed their initial liver buds protocol and, as stated previously, lack reproducibility [8]. In this for massive production and reproducibility making it context, improvements in bioprocess design and defin- ready for further GMP adaptation in order to scale up ition of strategies in compliance with quality and regula- production in GMP facilities for transplantation pur- tory guidelines are needed, being crucial the use of poses [12]. Moroever, the production of cells for human chemically defined serum-free culture media and the es- application greatly benefits from the use of fully defined, tablishment of robust specifications of starting materials xeno-free conditions rather than media supplemented and the critical quality attributes (CQA) of final product with sera, thus improving reproducibility and preventing [9, 10]. This can only be understood considering hPOs xeno-mediated infection or immune rejection when as a cell-based medicinal product, from a pharmaceutical transplanted into patients. production perspective, in compliance with current The generation of hPOs from the human pancreas is GMP guidelines. feasible, although not at the scale necessary to result in sufficient number of cells for clinical use [13]. In their Quality compliance in the production of organoids recent paper, Dossena and collaborators fixed a series of GMP in cell therapy refers to a system for ensuring that in-process controls and improved media formulation by medicinal products are consistently manufactured and replacing R-spondin-1 (RSPO1) conditioned medium controlled according to quality standards, thus minimiz- (CM) with recombinant RSPO1 protein, resulting in a ing the risks involved in the production process that GMP-compliant protocol intended to deliver a cryopre- cannot be eliminated otherwise through testing the final served hPO for further use in allogeneic therapies for drug product, so they are safe to be used in patients. To type 1 diabetes [14]. achieve this, GMP covers all aspects of production (e.g., Similar to other cell-based therapies, Dossena and col- starting materials, facilities, and equipment), personnel laborators paid special attention to define the criteria of (e.g., training, hygiene of staff), and documentation (e.g., acceptance of the starting materials in order to minimize standard operating procedures and registers to prove the risk of transmittable viral diseases and to ensure the Table 1 Quality product profile for hPO. Critical quality attributes of hPO and acceptance proposed by Dossena and collaborators that can be adapted in other developments of organoid production in accordance with pharmaceutical quality standards. Additional controls may include determination of endotoxins, mycoplasma, and sterility test according to the pharmacopeia. Ab antibody, Ag antigen, HBc hepatitis B core, HBV hepatitis B virus, HCV hepatitis C virus, HIV human immunodeficiency virus, VDRL-TPHA venereal disease research laboratory test-treponema pallidum hemagglutination test In process controls Acceptance criteria Donor Serology (HIV, HBV, HCV, anti-HBc, VDRL-TPHA) Negative Risk factors, systemic infections, diabetes Absence Amylase levels Within 3 times the normal range Biopsy Transport 21 ± 4 °C hPO Isolation Within 36 h from biopsy Identity (using ductal, mesenchymal, hematopoietic and endothelial markers) Presence of ductal progenitors (PDX1+/SOX9+) Metabolism Normal glucose consumption and lactate production Viability ≥ 70% Potency Capacity to differentiate into insulin-secreting cells Sterility (BacTAlerT) Sterile Karyotype Normal Vives and Batlle-Morera Stem Cell Research & Therapy (2020) 11:72 Page 3 of 4 procurement of healthy pancreatic tissue for the pro- Authors’ contributions duction of hPO (Table 1). To do so, pancreas biopsies All authors wrote the manuscript. All authors revised the manuscript. Both authors approved the final version of the manuscript. are processed within 36 h from extraction from donors aged 18–65 years, beating heart and declared Funding brain death, known cause of death, negative for HIV, Work in JV’s laboratory is supported by the Spanish Cell Therapy Network HBV, HCV, anti-HBc, VDRL-TPHA, absence of risk (TerCel, expedient no. RD16/0011/0028), awarded by Generalitat de Catalunya as Consolidated Research Group (ref. 2017-SGR-719). Project PI19/ factors, systemic infections, and diabetes, amylase 01788 is funded by Instituto de Salud Carlos III and co-funded by European levels within three times the normal range [14]. Simi- Union (ERDF/ESF) - A way to build Europe. lar to the original small-scale research method, the Availability of data and materials GMP-validated production process resulted in hPOs Not applicable. with equivalent dimensions, growth kinetics, metabol- ism, and cellular composition. In the characterization Ethics approval and consent to participate of hPOs, the authors reported the presence of ductal Not applicable. cells and ductal progenitors but differentiation into Consent for publication endocrine cells producing insulin was not demon- Not applicable. strated in this work. Remarkably, viability rates were always above 75% after thawing thus demonstrating Competing interests the feasibility of generating a bank of cryopreserved The authors declare that they have no competing interests. organoids, which could be further differentiated, ei- Author details ther in vitro or in vivo, into insulin-producing cells, 1 Cell Therapy Service, Blood and Tissue Bank (BST), Barcelona, Catalonia, as already shown to be feasible by other authors [15]. Spain. 2Musculoskeletal Tissue Engineering Group, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, This is a key point that will need to be addressed Catalonia, Spain. 3Medicine Department, Universitat Autònoma de Barcelona next to ensure clinical success, provided that the (UAB), Barcelona, Catalonia, Spain. 4Core Facilities Program, Centre for potency of hPO generated in this work will rely on Genomic Regulation (CRG), Barcelona, Catalonia, Spain. their capacity to generate insulin and restore Received: 26 December 2019 Revised: 26 December 2019 normoglycemia. Accepted: 4 February 2020 References Outlook 1. Clevers H. Modeling development and disease with organoids. Cell. 2016; Although further research is required in order to evalu- 165(7):1586–97. 2. Dekkers JF, Berkers G, Kruisselbrink E, Vonk A, de Jonge HR, Janssens HM, ate the therapeutic benefit of organoids in the context of et al. 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