Towards manufacturing of human organoids

Journal Pre-proof Towards manufacturing of human organoids

Aswathi Ashok, Deepak Choudhury, Fang Yu, Walter Hunziker PII:

S0734-9750(19)30160-0

DOI:

https://doi.org/10.1016/j.biotechadv.2019.107460

Reference:

JBA 107460

To appear in:

Biotechnology Advances

Received date:

16 April 2019

Revised date:

8 October 2019

Accepted date:

10 October 2019

Please cite this article as: A. Ashok, D. Choudhury, F. Yu, et al., Towards manufacturing of human organoids, Biotechnology Advances (2018), https://doi.org/10.1016/ j.biotechadv.2019.107460

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© 2018 Published by Elsevier.

Journal Pre-proof

Towards Manufacturing of Human Organoids Aswathi Ashok1,1 [email protected], Deepak Choudhury2,1,* [email protected]., Fang Yu2 [email protected], Walter Hunziker1,3 1Institute

of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore 2Bio-Manufacturing Programme, Singapore Institute of Manufacturing Technology (SIMTech), Agency for Science, Technology and Research (A*STAR), 2 Fusionopolis Way, #08-04, Innovis, Singapore 138634, Singapore 3Department of Physiology, 2 Medical Drive, MD9, National University of Singapore, Singapore 117593, Singapore

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author.

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*Corresponing

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Co-first authors

Journal Pre-proof Abstract Organoids are 3D miniature versions of organs produced from stem cells derived from either patient or healthy individuals in vitro that recapitulate the actual organ. Organoid technology has ensured an alternative to pre-clinical drug testing as well as being currently used for “personalized medicine” to modulate the treatment as they are uniquely identical to each patient’s genetic makeup. Researchers have succeeded in producing different types of organoids and have demonstrated their efficient application in various fields such as disease modeling, pathogenesis, drug screening and regenerative medicine. There are several protocols for

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fabricating organoids in vitro. In this comprehensive review, we focus on key methods of

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producing organoids and manufacturing considerations for each of them while providing insights on the advantages, applications and challenges of these methods. We also discuss pertinent

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challenges faced during organoid manufacturing and various bioengineering approaches that can improve the organoid manufacturing process. Organoids size, number and the reproducibility of

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the fabrication processes are touched upon. The major factors which are involved in organoids

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manufacturing like spatio-temporal controls, scaffold designs/types, cell culture parameters and

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vascularization have been highlighted.

Keywords: Organoids, Organoids manufacturing, Organoids production, Stem cells, Embryoid bodies, 3D cell culture, Air-

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liquid-interface

Journal Pre-proof 1. Introduction Organoid technology has recently emerged as a cutting-edge tool for fundamental research (Lancaster, Madeline A. and Knoblich, Juergen A., 2014; Sato et al., 2009) as well as translational medicine (Bartfeld and Clevers, 2017; Fatehullah et al., 2016; Sato and Clevers, 2013). Animals have been excellent models for research, however, not all the drugs that clear pre-clinical animal trials succeed during clinical trials due to differences in animal and human physiology (Akkerman, 2017; Kamb et al., 2006; Kaushik et al., 2018; van der Worp et al., 2010). Organoids recapitulate the complexity of the actual human organs and could potentially

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complement animal models in pre-clinical trials (Akkerman, 2017; Kaushik et al., 2018; Weeber et al., 2017). Manufacturing organoids in large scale could benefit a variety of fields including

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disease modelling (Huch et al.; McCracken et al., 2014; Weeber et al., 2017), personalized drug

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screening (Huang et al., 2015; Skardal et al., 2016), pathogenesis (Huch et al.; Weeber et al., 2017), cancer research (Drost and Clevers, 2018; Drost et al., 2017) and regenerative medicine

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(Lancaster, M. A. and Knoblich, J. A., 2014; Taguchi et al., 2014; Takebe et al.). Organoids are defined as scaled-down, miniature versions of an organ generated in vitro in a 3D culture system

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that can recapitulate its in vivo microenvironment (Kaushik et al., 2018). Recently, the definition of organoid has been narrowed down to self-organizing 3D structures grown from stem cells that

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organs (Dutta et al., 2017).

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mimic the in vivo environment, architecture and multi-lineage differentiation of the original

Organoids could be produced from various sources such as pluripotent stem cells (Lancaster et

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al., 2013; Quadrato et al., 2017) (i.e) embryonic stem cells (Volkner et al., 2016), adult stem cells (Broutier et al., 2016; Sato et al., 2009), patient-derived induced pluripotent stem cells (Crespo et al., 2017; Lancaster et al., 2013), and induced pluripotent stem cells from a donor’s skin fibroblast cells, blood cells or other cell type (Wang et al., 2018). These organoids can then be stored in biobanks for regenerative medicine and fundamental research (Sato and Clevers, 2013). Though both spheroids and organoids are 3D structures made of cells, they are not the same . Spheroids are 3D structures formed under non-adherent conditions from cancer cell lines (Sutherland Rm Fau - McCredie et al.), whereas organoids are 3D cell aggregates that comprises of cells that are more organ specific in lineage that develop from stem cells or progenitor cells in the presence of extracellular matrix in a 3D cell culture medium where cells self-organize recapitulating the in vivo organ-formation process (Lancaster, Madeline A. and Knoblich,

Journal Pre-proof Juergen A., 2014).

Organoids unlike spheroids, have a higher order self organization/self

assembly that is more similar to the in vivo organ formation and hence serve as better in vitro models as they better mimic the complexity of an actual organ (Li, 2019; Merck, 2019). When transplanted into animal models, organoids can further develop and become vascularized (Mansour et al., 2018) The term “spheroid” is sometimes also used to refer to an intermediate stage of organoid growth (Crespo et al., 2017; Freedman et al., 2015; McCracken et al., 2014). Organoids have recently become popular in vitro 3D cell culture model in areas of developmental biology, drug screening, and disease modelling. Arora et al. in 2017 found that

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13% of spheroids developed into mature intestinal organoids and hypothesized that the key

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specification required in spheroid maturation is the morphological feature of the inner mass and diameter (Arora et al., 2017). They demonstrated that spheroids with a diameter greater than 75

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μm act as pre-organoids that later mature into mature intestinal organoids (Arora et al., 2017). A study by McCracken et al. showed that only a small fraction of the spheroids matured in

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organoids (McCracken et al., 2014). Currently, spheroids produced are either scaffold based

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(Hydrogels or inserts) / non scaffold based 3D cell aggregates. The study by (Arora et al., 2017) and (McCracken et al., 2014) opens up avenues for organoid research from a manufacturing

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point of view, where spheroids can be manipulated to develop organoids. With regards to the above mentioned studies, different techniques (Costa et al., 2016) to produce spheroids such as hanging drop method, hanging drop, microfluidic-based assembly, spinner flasks and liquid

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overlay method have the potential to be further manipulated to develop organoids in future,

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though currently there isn’t much work on this yet (Bello, 2019, Sep 26)

Organoid models have been developed for a number of organs and include intestinal organoids (Sato et al., 2009; Spence et al., 2011), cerebral organoids (Lancaster, M. A. and Knoblich, J. A., 2014; Lancaster et al., 2013), kidney organoids (Taguchi et al., 2014; Takasato et al., 2016), hepatic organoids (Mitaka, 2002), retinal organoids (Boucherie et al.; DiStefano et al., 2018; Eiraku et al., 2008; Tucker et al., 2014; Volkner et al., 2016), pancreatic organoids (Greggio et al., 2013; Hohwieler et al., 2017; Huang et al., 2015; Li, X. et al., 2014), lung organoids (Bals et al., 2004; Escaffre et al., 2016; Fessart et al., 2013; Nadkarni, R. R. et al., 2016; Pageau et al., 2011; Vaughan et al., 2006), colonic organoids (Crespo et al., 2017),

gastric organoids

Journal Pre-proof (McCracken et al., 2014), cardiac organoids (Nugraha et al., 2019; Stevens et al., 2009), thyroid organoids (Toda et al., 2002), prostate organoids (Gao et al.), saliva-secreting organoids(Ferreira et al., 2019), mammary gland (breast) organoids (Lee et al., 2007; Simian et al., 2001; Weaver et al., 1997), lingual organoids (Hisha et al., 2013), , placenta organoids (Vogt, 2019) and spinal organoids(Research, 2019). In an earlier study, it was observed that cells behave differently in 2D and 3D cultures (Weaver et al., 1997), this was suspected to be one of the main reasons for therapeutic drug failures in clinical trials for drugs tested using 2D models. The identification of Lgr5 gene and its significance as a biomarker for adult stem cells in many tissues marked the

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beginning of research with 3D cell cultures (Hsu et al., 1998). In 2008, Sasai et al. used the

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SFEBq technique to create 3D cerebral cortex tissue from pluripotent stem cells (Eiraku et al., 2008). The significant role of Lgr5 gene to give rise to crypt-villus structures in the absence of a

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non-epithelial (mesenchymal) cellular niche and eventually intestinal organoid in vitro was demonstrated by Sato et al. (Sato et al., 2009). Discovery of this intestinal culture system was a

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major breakthrough in the field of organoids. The study also inferred that organoids developed

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from a crypt or a single stem cell (Lgr5+ cells) are indistinguishable (Sato et al., 2009). The starting cell type and system conditions, endogenous and exogenous signals and the physical

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characteristics of the culture environment were identified as three main characteristic features that influence the successful formation of organoids in vitro (Rossi et al., 2018).

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Commercial production of organoids at a large scale still needs to be established, although a few biotech companies are starting to move in that direction (Chakradhar, 2016). This paper

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elaborates on the key methods of producing organoids in vitro that have the potential to be used as standard protocols for mass organoid manufacturing. Before highlighting the methods of organoid production, important clinical applications of organoids are summarized. 2. Clinical applications of organoids Organoids have opened remarkable opportunities in pathogenesis and disease modelling, drug screening and regenerative medicine. These are described in detail below. 2.1 Pathogenesis and Disease models Animals are common models for pathogenesis studies (Campo, 2002; Gold et al., 2006; Lazear et al., 2016). Recent studies have revealed the usage of organoids as an alternative to animal models for disease modelling (Hwang et al., 2016; Lancaster et al., 2013). There are three main

Journal Pre-proof techniques to introduce microbes into organoids to generate disease models: (a) Infecting the dissociated cells before 3D organoid formation (b) microinjecting into the lumen of the organoids and (c) addition of microbes to 2D cultures derived from organoids (Dutta et al., 2017). Disease models can also be created by genome editing in organoids using CRISPR/Cas9 technique to study the genetic basis of diseases. One such example is the creation of genetically modified kidney organoids [produced from human pluripotent stem cells (hPSCs)] to model genetic kidney disease using CRISPR_Cas9 genome editing demonstrated by Freedman et al.(Freedman et al., 2015). There are also patient-derived organoids that model diseases, these

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have applications in personalized medicine and drug screening (Noordhoek et al., 2016).

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Lancaster et al. succeeded in producing miniature cerebral organoids that model microcephaly by using patient-specific iPSCs and RNAi (Lancaster et al., 2013). Cerebral organoids have been

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modeled for microcephaly (Lancaster et al., 2013). This was also the first study to report human iPSCs-derived cerebral organoids. Cerebral organoids have also been used as disease models to

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study numerous other conditions such as seckel syndrome (Gabriel et al., 2016), zika virus

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infections (Cugola et al., 2016; Garcez et al., 2016; Lancaster et al., 2013; Qian et al., 2016) and autism spectrum disorders (Mariani et al., 2015). McCracken et al. came up with a robust method

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for generating human gastric organoids and demonstrated its use as a model for the pathogenesis of Helicobacter pylori infection (McCracken et al., 2014). Retinal organoids modelled for the study of glaucoma (Tucker et al., 2014) and intestinal organoids (Spence et al., 2011) produced

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with future prospects of being used in disease modelling to study gastrointestinal diseases

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(Watson et al., 2014). Lung organoids produced using the air-liquid interface culture method were used to model Nipah virus infection (Escaffre et al., 2016). Nadkarni et al. in their review explained lung organoid systems catered for lung development and diseases (Nadkarni, R. R. et al., 2016). Hohwieler et al. made pancreatic organoids directly from human iPSCs and used them as a model for the pathogenesis of cystic fibrosis and other pancreatic disorders (Hohwieler et al., 2017). Strange et al. developed human testicular organoids to study the pathogenesis of zika virus infection (Strange et al., 2018). Organoids from both Pluripotent stem cells (PSCs) (Finkbeiner et al., 2012) (Porotto et al., 2019) as well as adult stem cells (ASC) (Bartfeld et al., 2015; Bartfeld and Clevers, 2015; Castellanos-Gonzalez et al., 2013; Ettayebi et al., 2016) (Zomer-van Ommen et al., 2016) (Yin et al., 2015) also have a great potential use as models for infectious diseases as they enable host-pathogen studies.

Journal Pre-proof Organoids & Cancer research A major attribute of organoids that makes it significant in cancer research is the fact that organoids derived from patient cells mimic the tumor characteristics (Drost and Clevers, 2018). Since organoids are genetically stable they are used to study cancer and tissue homeostasis (Kaushik et al., 2018). Broutier et al. recently developed primary liver cancer organoids for drug screening and disease modelling (Broutier et al., 2017). There are numerous patient-derived organoids that help are extensively used for cancer research (Drost and Clevers, 2018). The following is a list of all the major patient-derived tumour organoids, developed to date used

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extensively in cancer research: Liver (Broutier et al., 2017), prostate (Gao et al., 2014), Colon

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(Sato, Toshiro et al., 2011), Gastrointestinal (Seidlitz et al., 2019; Vlachogiannis, Georgios et al., 2018), Lung (Nadkarni, Rohan R. et al., 2016), Breast (DeRose et al., 2013) and bladder tumour

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organoids (Lee et al., 2018) . Recent research found that combining tumour organoids with

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fibroblast and immune cells resulted in models that enable the study of tumour microenvironment, thereby improving the immune-oncology applications of tumour organoids

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(Tuveson and Clevers, 2019). There are a number of well-known organoid cancer biobanks that aid in cancer research and drug discovery such as organoid biobank for breast cancer organoids

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(Sachs et al., 2018) , colon (van de Wetering et al., 2015) , ovarian (Kopper et al., 2019) and

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2.2.Drug screening

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gastric cancer organoids (Yan et al., 2018).

Production of patient-derived organoids that closely resemble the original tumors are considered a major breakthrough, as they offer an excellent alternative to pre-clinical drug testing (Weeber et al., 2017). There have been attempts to build a large organoid biobank to facilitate the study of drug development and screening (Weeber et al., 2017). Vlachogiannis et al. showed that drug responses from patient-derived tumor organoids had a high degree of similarity to the in vivo response of the patient in the clinic (Vlachogiannis, G. et al., 2018). Colonic organoids generated from iPSCs of patients with familial adenomatous polyposis was used for drug testing by Crespo et al. (Crespo et al., 2017). Numerous mutations on the cystic fibrosis transmembrane conductance regulator (CFTR) were found to be a leading cause of cystic fibrosis in human

Journal Pre-proof (Dekkers et al., 2013; Weeber et al., 2017).Patient derived intestinal organoids have been used for testing of cystic fibrosis drugs(Noordhoek et al., 2016). 2.3 Regenerative medicine Organoids can potentially be used as an alternative for organ transplantation in the future. Cerebral organoids are miniature versions of brains developed in vitro, used to study the development and function of the brain (Lancaster, M. A. and Knoblich, J. A., 2014). Liver

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transplants are required to save patients suffering from chronic liver disorders resulting in liver failure (Huch M Fau - Boj et al.). Scientists are trying to use liver organoids as an organ

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transplant alternative (Huch M Fau - Boj et al.). Takebe et al. succeeded in generating a vascularized human liver by transplanting liver buds produced in vitro and growing it in mice

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(Takebe et al.). Taguchi et al. have successfully achieved transplantation of kidney organoid in

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adult mice (Taguchi et al., 2014) which, if successful in humans, could one day replace kidney dialysis or renal transplantation in kidney failure patients. Schwank et al demonstrated gene

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correction in cystic fibrosis patient-derived organoids using CRISPR/Cas9 (Schwank et al., 2013). All the aforementioned studies showcase the importance of organoids in regenerative

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medicine, however there many safety and compatibility issues linked with the application of organoids as transplants into humans (Fatehullah et al., 2016). The need for GMP compliant

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culture system for manufacturing organoids using scaffolds that are tissue compatible is

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necessary to reduce tissue rejection and organoid incompatibility issues (Dakhore et al., 2018). Types of Organoids (Based on source): Depending on the source, there are two main types of organoids produced (Huch and Koo, 2015)- a) Organoids produced from adult stem cells (ASC Approach): The source of these organoids are adult stem cells/progenitor cells or fragments of isolated primary tissues (Huch and Koo, 2015). This approach for organoid production mainly depends on the tissue repair/renewal principles. Wnt Signalling in adult stem cells have been identified as the key factor that enables the organoids produced from ASC approach expand indefinitely (Maimets et al., 2016). The crypt isolation protocol optimized by Sato et al. in 2009 is mainly used for generating intestinal organoids from primary tissues. To date, only epithelial organoids can be produced from adult stem cells such as

Journal Pre-proof intestinal organoids (Broutier et al., 2016; Ootani et al., 2009; Sato et al., 2009), gastric organoids (Barker et al., 2010; Bartfeld et al., 2015; Li, Xingnan et al., 2014; Stange et al., 2013), liver organoids (Broutier et al., 2016; Huch et al., 2013), pancreatic organoids (Broutier et al., 2016), salivary gland (Maimets et al., 2016; Nanduri et al., 2014), tongue (Hisha et al., 2013) b) Organoids produced from pluripotent stem cells (PSC Approach): The source for these organoids are either embryonic stem cells (ESCs) taken from inner cell mass of blastocysts or induced pluripotent stem cells (iPSCs) produced from somatic cells. This approach mainly involves directed differentiation of pluripotent stem cells (both ESCs as well as iPSCs) to form

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the respective stem cell progenitor cells. Definitive endoderm layer is what gives rise to digestive

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tracts, respiratory tract epithelial lining, lungs, thyroid, pancreas, thymus and liver. The appearance of the primitive streak in the posterior epiblast marks the beginning of the

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differentiation process (D'Amour et al., 2005). The ingression of posterior epiblast cells through the primitive streak leads to EMT (epithelial-to-mesenchymal transition) which later either

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become the mesoderm or definitive ectoderm (D'Amour et al., 2005; Shook and Keller, 2003). It

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was found that disruption in WNT or TGFβ signaling pathway prevented the appearance of the primitive streak, mesoderm or definitive endoderm (D'Amour et al., 2005; Haegel et al., 1995;

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Liu et al., 1999). Using this knowledge, various methods were developed to edit the molecules involved in signaling pathways to direct differentiation of stem cells and form organoids using

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Matrigel as the scaffold (D'Amour et al., 2005; McCracken et al., 2014; Spence et al., 2011).

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The growth and development of organoids produced from PSC approach is difficult and they also do not proliferate indefinitely hence researchers try and transfer it to ASCs approach whenever possible (Spence et al., 2011; Volkner et al., 2016). The protocol developed by Spence et al. involves the formation of intestinal organoids by PSC approach. Beginning with the formation of definitive endoderm by directed differentiation, a method developed by D'Amour et al. (D'Amour et al., 2005). Spence et al. used a three-day Activin A protocol (Spence et al., 2011). For hindgut differentiation, the definitive endoderm cells were incubated for 4 days in 2% FBS-DMEM/F12 media containing FGF4 and Wnt3a (Spence et al., 2011). 3D floating spheroids were formed by treatment with growth factors followed by further culturing the intestinal organoids using a crypt isolation protocol as described in (Sato et al., 2009). They further modified the process by manipulating a series of growth factors to re-create the

Journal Pre-proof embryonic intestinal development in vitro (Spence et al., 2011). Similarly generating intestinal organoids from human PSCs involves the formation of definitive endoderm by a directed differentiation method as described by D'Amour et al. in 2005 (Gao et al., 2014). This is followed by floating spheroid formation, which is then embedded into Matrigel leading to the formation of the intestinal organoid using the intestinal culture system demonstrated by Sato et al. in 2009. Yet another successful work is production of retinal organoids by Völkner et al., where after culturing human ESCs and embedding it in Matrigel, a neuroepithelium trisection protocol to retinal organoid (Volkner et al., 2016). All the organoids produced from pluripotent

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stem cells ( ESCs & iPSCs) are as follows (Huch and Koo, 2015): organoids from endoderm

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include intestinal (Spence et al., 2011) ,liver (Takebe et al., 2013), gastric (McCracken et al., 2014), lung (Dye et al., 2015) and thyroid (Antonica et al., 2012) organoids from ectoderm-

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cerebral organoids (Lancaster et al., 2013; Muguruma et al., 2015), inner ear (Koehler et al.,

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2013) and retina organoids (Eiraku et al., 2011; Eiraku et al., 2008; Volkner et al., 2016).

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3. Current methods for the generation of organoids This section focuses on the common methodologies employed in the generation of organoids

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(Figure 1, Table 1). The advantages and disadvantages of each method and considerations for organoids manufacturing are discussed. Understanding of the limitations of these methods for an

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organoid generation will help researchers to design more refined and reliable processes for manufacturing organoids at a larger scale. All things considered, the current organoid culture

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systems can be broadly be categorized into four main categories (i) suspension culture system for organoids, (ii) organoid formation using ECM (Matrigel) scaffolds (iii) methods using spinning bioreactors, and (iv) air-liquid interface (ALI) methods. 3D Culture System for Organoids 3.1. Suspension culture system for organoids Morizane et al. developed an easy and efficient monolayer culture protocol to mass-produce kidney organoids within 12 days from nephron progenitor cells (hPSCs to NPSCs differentiation takes 7-9 days) by using 96 well round bottom ultra-low attachment plates and chemically defined compounds (Figure 2). The main advantage of this method is that this protocol can be

Journal Pre-proof used to produce both 2D and 3D kidney organoids. However, for detailed study of disease and phenotype 3D system cultured organoids are recommended (Morizane and Bonventre, 2017). This protocol allows the researchers to examine the cells of the kidney organoid at each stage of differentiation, thereby greatly widening the future possibilities of studying inherited kidney diseases (Morizane and Bonventre, 2017). Daquinag et al. used a magnetic levitation method using nanoparticle assembly to create 3D white adipose tissue growth and development in organoids (Daquinag et al., 2012). Tseng et al. first demonstrated the assembly of 3D co-culture structure of bronchiole using four cell types:

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fibroblast cells, endothelial cells, epithelial cells and smooth muscle cells using magnetic

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levitation (Tseng et al., 2013). Ferreira et al. demonstrated the production of saliva-secreting organoids by a magnetic levitation method and used it to repair salivary gland hypofunction

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(Ferreira et al., 2019). A commercially available magnetic levitation based culture system has been used to culture 3D organoids using nanoparticles in 24 well and 96 well bio-assembler

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plates (Souza et al., 2010)

In magnetic levitation approaches, cells are cultured close to confluence followed by a

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detachment of cells from the bottom of the culture flask by trypsinization. The cells are counted and added into 24 well or 96 well plates. The cell suspension is diluted if needed. It is then treated with Nanoshuttle-PLTM reagent by adding and dispersing the Nanoshuttle-PLTM reagent

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over the cells, thereby introducing biocompatible magnetic nanoparticles to cells. Culture

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Isolation lid is placed on top of the plate along with a magnetic drive assembly lid containing Nanoshuttle-PLTM treated cells. The bio-assembler is then placed in the incubator. Within a very short time the cells get levitated to the air-liquid interface and get concentrated under the magnets leading to the development of 3D cellular structure (Souza et al., 2010).

Advantages of magnetic cell levitation culture system: Prompt cell-cell interactions and development of 3D structures that mirror the in vivo tissue representation. Magnetic Bio-assemblers are compatible with primary and stem cells, different media recipes, standard culturing protocols, and diagnostic techniques.

Journal Pre-proof 3.2 Organoid formation using ECM (Matrigel) scaffolds 3.2.1 Crypt isolation method for organoid production Most of the protocols to produced organoids from adult stem cells involves this method. Clevers and team (Sato et al., 2009) were the first to optimize an efficient culture system to generate intestinal organoids using Matrigel as the extracellular matrix (ECM) from adult Lgr5 stem cells (Figure 3 & Figure 3A). This intestinal organoid culture system is commonly called the RSpondin Method (Fatehullah et al., 2016; van de Wetering et al., 2015). Matrigel is the trade

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name of the gelatinous protein mix produced by the sarcoma cells of Engelbreth-Holm-Swarm

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(EHS) mouse which resembles the ECM surrounding most living cells in vivo, used by researchers as ECM support for the in vitro culturing of cells (Benton et al., 2009; Benton et al.,

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2011; Hughes et al.). The development of the intestinal culture system was a major breakthrough

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in the field of stem cells. Sato et al. demonstrated that a single Lgr5 stem cell present at the tip of the intestinal crypt can self-organize into a crypt-villus structure and form intestinal organoids

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without the support of mesenchymal tissue. Crypts are the deep pit that extends inwards into the connective tissue at the margins of the small intestine.

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In this procedure, isolated crypts from the small intestine of mice are incubated for 30 min at 4℃ in PBS solution with EDTA. Polymerization of crypts with Matrigel is a major step in this After

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procedure. Crypts are then mixed with Matrigel and plated in 24 well plates.

polymerization of Matrigel crypt culture medium, Advanced DMEM/F12 from Invitrogen is

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added. The crypt culture medium, Advanced DMEM/F12 contains growth factors R-spondin 1, EGF, and Noggin (Sato et al., 2009). Organoids obtained can be preserved for a long duration by freezing in liquid nitrogen and then storing at -80℃ (Xue and Shah, 2013).

Sato et al. further modified the culture medium in this protocol to produce long-term expanding intestinal epithelial organoids from human colon, adenoma and adenocarcinoma (Sato, T. et al., 2011). The signaling molecules such as Wnt, TGF-⍺, EGF and Notch ligand Dll4 produced by the CD45+ Paneth cells have a significant role in the maintenance of the Lgr5 stem cells, emphasizing the significant role of co-culturing Paneth cells with Lgr5 stem cells for improved organoid production (Sato et al., 2010). In 2016, O’Rourke et al. optimized a protocol adapted

Journal Pre-proof from the crypt isolation protocol of Sato et al., 2009 and used it to isolate, culture and maintain mouse intestinal stem cells (O'Rourke et al., 2016).

Advantages and limitation of the crypt isolation method Most of the method using Matrigel as a scaffold for organoid formation use protocols that are modified versions of crypt isolation organoid culture system by Sato et al. The major advantage of this method is its simplicity (Sato et al., 2009) and it is also an established long-term culture system for organoid culture (Sato et al., 2009). Secondly, this method can be used to produce

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organoids from multiple sources such as adult tissue (Sato et al., 2009), fetal tissue, ESCs, and

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iPSCs (Fatehullah et al., 2016).

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The primary issue using this method is the difficulty in recapitulating the in vivo signaling and growth factor gradients in the Matrigel matrix. The possible solutions to this limitation are to

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create a concentration gradient using microfluidic technologies (Fatehullah et al., 2016).

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Secondly, since organoids produced using this method depend on Matrigel which is mouse sarcoma produced ECM, these organoids are not compatible with human organ transplantation

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(Fatehullah et al., 2016).

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3.3. Organoid formation via embryoid bodies formation and agitation using spinning bioreactor The embryoid bodies formation has been mainly used to produce cerebral and retinal organoids (Figure 4). Recreating human brain disorders in animal models have always been difficult. Lancaster et al. tried to generate human cerebral organoid as a disease model for neural disordermicrocephaly and check if the patient-derived cerebral organoid could act as an alternative model for animal models to study neurodevelopmental disorders (Lancaster et al., 2013). Lancaster et al. reprogrammed skin fibroblast cells of a patient with microcephaly using lentiviral delivery of the four major reprogramming factors, Oct 4, Sox2, Klf4 and c-Myc to produce induced pluripotent stem cells (Lancaster et al., 2013). They succeeded in optimizing a cerebral organoid culture system to produce both mouse and human cerebral organoids in vitro from pluripotent stem cells. When pluripotent stem cells form organoids, cells form aggregates

Journal Pre-proof in some cases as an intermediate stage. These 3D cell aggregates of pluripotent stem cells are called embryoid bodies, observed especially in the formation process of cerebral organoids. The human cerebral organoids produced emulate earlier stages of the human brain. Mitomycin C growth inactivated MEFs were used to culture mouse ESCs and passaged using standard protocols. As described by Eiraku et al. the cells were then trypsinized and plated in each well of an ultra-low binding 96 well plate in a differentiation medium(Eiraku et al., 2008). Embryoid bodies formed using this protocol (Koike et al., 2005) were then transferred into neural induction medium on day 5, the neuroectodermal tissues were then embedded in Matrigel droplets on day

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11 and transferred to the spinning bioreactor after 4 days of stationary growth inside Matrigel

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(Lancaster et al., 2013). ESCs of the mouse which, in contrast to those from human divide at a faster rate, were transferred to neural induction medium on day 4, embedded in Matrigel droplets

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on day 6, and on day 9 transferred to the spinning bioreactor (Lancaster et al., 2013). Lancaster et al. published a detailed cerebral organoid protocol and discussed the challenges faced during

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the process (Lancaster, M. A. and Knoblich, J. A., 2014).

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Quadrato et al. in 2017 further modified the protocol developed by Lancaster et al. and demonstrated the possibility of developing cerebral organoids with mature features for extended

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periods by controlling the neuronal activity within the organoids. This can be done using light stimulation of photosensitive cells (Quadrato et al., 2017). Wang et al. developed a protocol

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combining both stem cell and organ-on-a-chip technology in 2018 (Wang et al., 2018). Recent studies show improvement in producing mature cerebral organoids (Giandomenico et al., 2018;

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Quadrato et al., 2017). DiStefano et al. used a rotating wall vessel bioreactor to produce 3D retinal organoids (DiStefano et al., 2018). Cerebral organoids (Giandomenico et al., 2018; Lancaster et al., 2013; Quadrato et al., 2017) and retinal organoids (DiStefano et al., 2018) are produced using this method that involves embryoid body formation in the process. Advantages and limitation of the embryoid body method The use of a spinning bioreactor in addition to Matrigel scaffold tackles one of the major challenges in organoid manufacturing, namely optimal nutrient and oxygen supply to the cell in the organoid. This is achieved by the regular supply of media by the spinning bioreactor (Lancaster et al., 2013). Moreover, cerebral organoids (Lancaster et al., 2013) produced using the protocol optimized by Lancaster et al. can be stored up to over 1 year (15 months) in the

Journal Pre-proof spinning bioreactor, though the organoids start to shrink after 6-7 months (Lancaster et al., 2013). The cerebral organoids produced using this protocol not only exhibit the rudimentary mammalian neural development but epitomize human brain development (Lancaster et al., 2013). Furthermore, Lancaster et al. succeeded in modelling these cerebral organoids with the disorder “microcephaly” from patient-derived iPSCs, which is advantageous because this particular disorder has been extremely difficult to model in mice (Lancaster et al., 2013). The major issue using the “embryoid bodies” based approach is the lack of vascularization (Ling et al., 2019). Addition of a spinning bioreactor to the production process to ensure constant

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supply of media so as to meet the need of nutrient and oxygen of the cells in the organoids has

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helped to overcome this problem but only up to an organoid size of approximately 4mm (in diameter) (Lancaster, M. A. and Knoblich, J. A., 2014). The cerebral organoids produced using

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this method mimic the initial human brain development but fails to show tissue maturation and

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complexity as seen in the adult brain (Lancaster et al., 2013). Secondly, the neural and nonneural tissue cross-talk is limited in the organoid as it lacks the surrounding embryonic tissues

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(Lancaster, M. A. and Knoblich, J. A., 2014). Organoid size is another limitation in this method. The organoids can only reach up to a size of 4mm in diameter due to the lack of nutrient

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exchange and limited oxygen exchange at the center of the organoids (Lancaster et al., 2013)

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3.4 Air liquid interface (ALI) methods for organoid culture Air liquid interface is a method in which the top layer of the cultured cells is exposed to air, and

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the basal surface is in contact with the liquid medium (Figure 5). The organoids are cultured in a gel and are directly exposed to air instead of submerged in a culture media. Tado et al. first used the air- liquid interface method on thyroid tissue in 2002 (Toda et al., 2002). Inspired by previous work, Ootani et al. developed a protocol to produce intestinal organoids (Ootani et al., 2009). Studies by Fatimah et al. revealed that the air liquid interface method could be used to produce human amnion epithelial cells (HAECs) as it stimulates an early differentiation of HAECs. They demonstrated its potential to be used as a substitute for skin regeneration (Fatimah et al., 2013). In 2014, Takasato et al. optimized a protocol using air liquid interface method to direct the process of embryonic stem cell differentiation into formation of a self-organizing structure including formation of nephron in vitro, by synchronizing the induction of the progenitors for both the collecting duct (UB-Ureteric bud) and nephron (MM-Intermediate

Journal Pre-proof mesoderm-derived metanephric mesenchyme) (Takasato et al., 2014). Takasato et al. generated kidney organoids that matched the first trimester human kidney with each kidney organoid containing about 500 nephrons (Takasato and Little, 2015). A detailed protocol that simultaneously induces all four progenitors (nephron progenitors, endothelial progenitors, ureteric epithelial progenitors and renal interstitial progenitors) to generate human kidney organoids from human pluripotent stem cells was developed in 2016 (Takasato et al., 2016). This is considered to be a major landmark in the research on kidney organoids because previously it was a major challenge to induce all four progenitors simultaneously. Li et al. demonstrated that

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a single air liquid interface could model several gastrointestinal malignancies from stomach,

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colon and pancreas in mesenchymal and primary epithelial organoid culture without the need for any additional growth factors. They also extended their study to successfully produce embryonic

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pancreatic organoids within a short period of time (Li, X. et al., 2014). Li et al. optimized a collagen-based ALI long-term culture methodology to expand gastrointestinal epithelium tissue

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and produce intestinal organoids. Usui et al. produced normal and tumor colorectal tissues from

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human patient tissues using ALI organoid culture system (Usui et al., 2018). Takasato et al. used an air liquid interface method to produce kidney organoids (Takasato et al.,

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2016). Calvin Kuo lab, Stanford University used the ALI method to produce gastric organoids (Li et al., 2016), intestinal organoids (Ootani et al., 2009), and pancreatic organoids. This method has also allowed them to produce cancer organoids. ALI method is currently mainly used to

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produce kidney organoid (Rossi et al., 2018), cerebral organoid (Giandomenico et al., 2018),

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lung organoid (Bals et al., 2004; Fessart et al., 2013; Pageau et al., 2011; Vaughan et al., 2006) and skin organoids (Fatimah et al., 2013).

Advantages and limitations of ALI method The main advantage of the ALI organoids is that they accurately replicate the original organ in terms of physiology, multilineage differentiation and organ structure (Usui et al., 2018). Interfollicular epidermal cells grow on the basement membrane and are exposed to air in vivo, using this knowledge Pruniéras et al. cultivated keratinocytes with a single air liquid interface (Pruniéras et al., 1983). Later it was found that organoids produced using ALI culture method were more precise as it accurately recreates the mode of cell growth that occurs in vivo as this leads to a complete differentiation (Pruniéras et al., 1983; Usui et al., 2018). Cell-stimulant

Journal Pre-proof interactions and cell-cell interactions are better in cells cultured using ALI compared to submerged cell culture systems (Upadhyay and Palmberg, 2018). ALI (Ootani et al., 2009) enables enhanced oxygen transport from both air-collagen interface(top) and semi-permeable membrane interface (bottom) resulting in a higher oxygen concentration within the system compared to the submerged culture system (Sato et al., 2009) which only allows oxygen transport from the top layer of the submerged culture media (DiMarco et al., 2014). A study by DiMarco et al. also showed that oxygen gradient within submerged cultures was 1.5 times higher than that in ALI culture system (DiMarco et al., 2014). This improved oxygenation in the ALI

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organoid cultures helps preserve the organoids for a longer period of time (Ootani et al., 2009)

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and also accelerates self organization in organoids (Sekiya et al., 2019). Culturing cells (keratinocytes) first under submerged conditions for 7 days to form the epidermal layer

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(monolayer) followed by culturing the cells in ALI culture system to further promote progressive differentiation has proven to be efficacious even in the process of 3D bioprinting of human skin

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which resulted in the formation of a multi-layered 3D skin construct (Epidermis, dermis and

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stratum corneum) (Lee et al., 2013). Microfluidic device “human lung-on-chip” with integrated ALI culture system provides improved ways to study drug efficacy and toxicity(Huh et al.,

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2011). ALI method has also been effective in creating patient-derived immunotherapy organoid models that retain its native tumour microenvironment (Neal et al., 2018)

.This is a major

breakthrough as the previous co-culture methods to emulate tumour microenvironment by

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combining tumour epithelia with additional cellular components such as tumour infiltrating

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lymphocytes were extremely elusive (Dart, 2019; Neal et al., 2018). Though ALI method has many advantages over other culture systems, it is not without challenges- Despite the successful production of kidney organoids by Takasato et al., they failed to produce kidney organoids that mature to the adult stage with ALI method (Takasato et al., 2016). ALI culture system is used to culture mainly epithelial cells and to develop organoids containing epithelial cells. Use of epithelial cells for culture in ALI culture system is challenging as there are chances of contamination by microbial or fibroblast cells. Also, use of primary epithelial cells from animals for maintaining the culture in ALI system involves sacrifice of a myriad number of animals compared to methods that use an already established cell line (Delgado-Ortega et al., 2014). Recently cerebral organoids (Giandomenico et al., 2018) were developed using ALI method, this opens up the use of this culture system to develop organoids of other cell type as well. However,

Journal Pre-proof currently most of the organoids developed so far using this culture system are those developed from epithelial cell cultures. 3.5 Recent advances in organoid research- 2D Monolayer culture systems of organoids

Cells in 2D culture systems grow by adhering to the base of the culture flask, petri dish or transwells, mostly growing into monolayer cultures (Sun et al., 2006). In 2018, Thorne et al. demonstrated that dissociated 3D organoids (formed using crypt isolation culture system demonstrated by Sato et al. (Sato et al., 2009)) can produce 2D enteroid monolayer (Thorne et

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al., 2018). They used 96 well plates coated with thin layered Matrigel to optimize a scalable

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method to culture 2D enteroid (or organoids recapitulating the small intestine (Foulke-Abel et al., 2016)) monolayers that emulates some of the important features of 3D organoids as well as in

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vivo conditions of an organ (Braverman and Yilmaz, 2018; Thorne et al., 2018). 2D Monolayer culture of organoids has a wide variety of applications such as intestinal epithelial

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barrier models to study the dynamics of host-microbe interactions, as well as molecular

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mechanisms involved in differentiation and function of different types of IECs (Intestinal Epithelial cells) (Nakamura, 2019). These monolayer cultures also permit high throughput

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microscopy for further study of the organoids (Thorne et al., 2018). A similar study by Hee et al. generated 2D monolayer cultures by enzymatic dissociation of 3D porcine intestinal organoids (van der Hee et al., 2018). In this study, 5-day spheroid cultures

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were disrupted into a single cell suspension and plated in Matrigel coated culture plates or

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transwells to produce monolayers (van der Hee et al., 2018). This study also demonstrated that cell seeding densities directly affected the capacity to form a monolayer (van der Hee et al., 2018). Furthermore, Liu et al. established a 2D culture system to support self-sustaining of Lgr5 intestinal stem cells monolayer. It was also found that 10μm thick Matrigel greatly supported Lgr5 ISCs (Intestinal Stem Cells) monolayer culture while thicker Matrigel layers (50 μm) supported 3D organoid development (Liu et al., 2018). Though one major limitation of using 2D monolayer cell culture of organoids for studies is the difficulty in passaging (Braverman and Yilmaz, 2018).

Journal Pre-proof 4. Challenges in organoids manufacturing and possible answers Organoids are a thriving topic of research with numerous clinical and pre-clinical applications, with the scientific community exploring ways to overcome issues that currently limit their full potential. One of the biggest challenges to organoid manufacturing is its limited reproducibility, which possesses a major hurdle if organoids are to be used for toxicity testing or another high throughput testing in future (Kelava and Lancaster; Lancaster et al., 2013). Organoid production scalability, batch to batch variation, cellular composition and architecture of organoids are all essential factors that affect the reproducibility of data obtained with organoids (Nugraha et al.,

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2019). In a recent study, Krefft et al. succeeded in generating a standard procedure to produce

sample to sample variability (Krefft et al., 2018).

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reproducible forebrain type cerebral organoids from human iPSCs which demonstrated improved

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Another important limitation is non-vascularization (Kaushik et al., 2018). Issues of

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vascularization could be potentially solved by either using a spinning bioreactor or by coculturing with endothelial cells (Kaushik et al., 2018). Insufficient supply of nutrient and oxygen

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exchange to the center of the organoids is the reason why most healthy tissues are at the periphery of the organoid, affecting the overall tissue patterning(Kelava and Lancaster; Lancaster

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et al., 2013). To improve the tissue vasculature, Allenby et al. fabricated ceramic hollow fiber constructs in 2018 (Allenby et al., 2018). Mansour et al. remarkably succeeded in producing

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vascularized human brain organoids in the mouse brain in vivo (Mansour et al., 2018). This was a major breakthrough as it eliminates most of the challenges faced due to limited vascularization

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of organoids (Mansour et al., 2018). This technique of cerebral organoids transplantation into mouse brain method definitely eliminates some of the problems/limitations faced by the in vitro culture systems (Lancaster, 2018), but faces ethical issues. A third major hurdle for manufacturing of organoids is tissue maturation (Kaushik et al., 2018). In most cases, early stages of development and organization in the organoids are well developed, but the tissues fail to evolve into a functionally developed and mature organ (Kaushik et al., 2018). Albeit, studies show that intestinal organoid produces Lgr5 stem cells that indicate further maturation (Sato et al., 2009). Giandomenico et al. have successfully optimized an efficient protocol for long-term production of mature cerebral organoids using ALI (Giandomenico et al.,

Journal Pre-proof 2018). In the case of generation of cardiac organoids, the role of maturity of the cardiomyocytes cells used in the co-culture is yet to be discovered (Nugraha et al., 2019). The absence of continuous anterior-posterior or dorsal-ventral axis that guide and support proper organoid directionality which results in the heterogeneity of organoids produced is another confounding issue, especially for cerebral organoid, (Kelava and Lancaster). Various parts of the cerebral organoid are randomly arranged when compared to the actual brain organ (Bagley et al., 2017). Knoblich and his team have been working on solving this limitation via two approaches: (a) by combining bioengineering techniques along with organoids to rectify the positioning of

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dorsal-ventral axis and anterior-posterior axis of the brain using biocompatible bioengineered

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fibers (PLGA fibers) poly (lactic-co-glycolic acid) and (b) by producing each distinct regions of the brain separately in vitro and recapitulating the exact order in the actual brain. Bagley et al.

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succeeded in producing the dorsal-ventral axis of organoids by fusing organoid identities of

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dorsal and ventral forebrain, emphasizing on the significance of such fused cerebral organoid cultures in modeling complex interactions between different parts of the brain (Bagley et al.,

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2017). The most recent studies showed successful assembling of brain organoids by rectifying the dorsal-ventral axis and posterior-anterior axis positioning. These so-called “assembloids” are

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considered to be the next generation brain organoids (Paşca, 2019). Biological complexity is another challenge for organoid manufacturing. Tumors in our body

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display complex genetic heterogeneity. Tumoroids are created in vitro to resemble the original

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tumors, although the extent to which the tumoroids hold the complexity and possible changes of the original tumors is still unclear (Dutta et al., 2017). Organoids produced using rigid extracellular matrices (ECM) are limited in their usage in drug screening as the ECM can hamper drug penetration (Fatehullah et al., 2016). One of the major limitations of organoids is the lack of blood cells, stroma and immune cells (Drost and Clevers, 2018). Bioengineering techniques are being used to incorporate additional cellular elements to overcome this problem (Yin et al., 2016). Yet another success in research is the immunotherapy models (patient-derived organoid models) that retain the tumour microenvironment produced by ALI method. This could open up great possibilities for in vitro modelling in personalised immunotherapy for cancer research (Dart, 2019).

Journal Pre-proof Organoids produced using Matrigel (which is a mouse sarcoma cell product) as the extracellular matrix was observed to cause compatibility issues in humans restricting the use of organoids in human organ transplantation (Fatehullah et al., 2016). Currently, research focusing on the development of a better defined ECM that abide by human regulations of organ transplantations are being carried out (Fatehullah et al., 2016). Furthermore, organoid production relying on embedding the stem cells into Matrigel restricts the surface to mass ratio, thereby limiting the manufacturing of organoids in a large scale (Lu et al., 2017). Lu et al. demonstrated a new capsule-based organoid production method that overcomes this limitation and produces

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organoids at a large scale with a better cell recovery (Lu et al., 2017). These capsules are

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produced using a two fluidic electrostatic co-spraying method and the core of the capsule consists of Matrigel that supports the growth of the organoids.

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Size of organoids is a crucial factor that restricts organoid technology from reaching its full potential and limiting their manufacturing. The maximum distance that oxygen and nutrients can

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diffuse inside the organoid determines the size of the organoids in culture (Akkerman, 2017). As

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the size of the organoids increases, an oxygen gradient is created within the organoids which ultimately leads to limited availability of oxygen to the center of organoids causing death in the

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central part of organoid after it reaches a particular size (Skardal et al., 2016) thereby limiting the production of organoids of larger sizes (Akkerman, 2017). Mathematical models of cerebral organoids shows that 1.43mm is the maximum attainable size of cerebral organoids (Akkerman,

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2017), although researchers have succeeded in generating cerebral organoids with a size up o

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4mm, with some cell death been observed at the center of the organoids (Akkerman, 2017; Lancaster, M. A. and Knoblich, J. A., 2014; Lancaster et al., 2013; McMurtrey, 2016). Moreover, Takasato et al. managed in generating larger sized kidney organoids (up to 6mm in diameter) by using an ALI method. (Takasato et al., 2016). These are exceptional cases with an average organoid size of 3mm commonly being obtained (Akkerman, 2017; Eicher et al., 2018). Recently, a very intriguing research carried out by Dr Alysson Muotri and team, where they developed a pea sized Neanderthal brain organoid. These Neanderthal organoids unlike its human organoid counterpart differs in size , appearance and neuronal activity (Cohen, 2018). In order to consider large-scale manufacturing of organoids, the standardization of size may be crucial (Eicher et al., 2018). Organoid imaging is an additional issue faced by researchers which

Journal Pre-proof are being overcome with deeper imaging and high throughput screening imaging techniques especially in the case of cardiac organoids (Nugraha et al., 2019).

5. Technologies to improve organoids manufacturing Combining bioengineering tools with 3D cell culture techniques definitely help in improving the size of the organoids formed and eliminates a number of challenges currently faced (SaglamMetiner et al., 2019). Factors to be considered to improve manufacturing of organoids are as follows (Figure 6): (a) scaffolds (b) regulating culture parameters such as oxygen distribution,

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cell-cell interaction, pH level etc. (c) spatiotemporal control of signaling and (d) efficient

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vascularization.

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Scaffolds: Most of the organoid culture system uses scaffolds made of Matrigel. But as described earlier Matrigel is incompatible with humans hence hinders with transplantation into humans.

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Alternative functional scaffolds made of biomolecules such as basic fibroblast growth factors

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(BFGFs), vascular endothelial growth factors (VEGFs) and platelet-derived growth factors (PDGFs) have improved the vascular networks of organoids produced (Yin et al., 2016). Defined

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ECM which include alternatives such as biomimetic scaffolds made from both natural and synthetic polymers (polyethylene glycol and polyacrylamide) is being developed to overcome the issues caused due to Matrigel in organoid research (Fatehullah et al., 2016; Yin et al., 2016).

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Regulating culture parameters: Sensors and devices that cause minimal disturbances to the in

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vivo cell environment and the natural processes can enable precise monitoring of these cell parameters (Yin et al., 2016), for example by direct in vivo measurements of oxygen tension using two-photon phosphorescence lifetime microscopy (Spencer et al.). Control over cell-cell interactions was made possible by using silicone substrate microscale devices (Hui and Bhatia, 2007). Microfluidic organs-on-a-a-chip also allows precise control and measurements on these important cell parameters, but they do have their own limitations (Bhatia and Ingber, 2014; Yin et al., 2016). Spatio-temporal control: The difficulty in recreating in vitro scaffolds that replicate the spatialtemporal signals to cells is the main cause of the observed difference between the organogenesis in vivo and in vitro (Yin et al., 2016). Recent advances envision ways to overcome this limitation by using light-mediated patterning to manipulate the ECM (Yin et al., 2016). Use of a more

Journal Pre-proof defined ECM as an alternative to Matrigel in organoid culture is crucial for improved spatiotemporal cues (Yin et al., 2016) and tissue regeneration applications. Efficient vascularization: Researchers have focused on several different bioengineering approaches to gain more control over the production of organoids and their behavior (Yin et al., 2016). For the proper development of functional organoids, in vitro vascularization is very important in order to provide a sufficient supply of nutrients and oxygen (Yin et al., 2016). One approach to achieve efficient vascularization is to seed the endothelial cells within the system to allow for neo-angiogenesis to occur. Alternatively, scaffold-based methods in which 3D

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microfluidic devices are used to allow uniform flow and mass transfer could be engineered. Yet

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another bioengineering approach could take advantage of bioprinting to seed the channels with vascular cells (Kang et al., 2015; Zhang et al., 2016). Bioprinting could be used to generate

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complex multicellular structures by layering different cell types along with hydrogel-based bioinks in a 3D bioprinter (Choudhury et al., 2018b; Kang et al., 2015). Recent studies showed

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improved kidney organoid vascularization with the application of fluid flow across the surface of

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partially differentiated kidney organoids attached to extracellular matrix housed within a 3D printed millifluidic chip (Allison, 2019; Homan et al., 2019). Later studies showcasing how this

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technique could help study polycystic kidney diseases was demonstrated (Boettner, 2019, Feb 11). Lancaster et al. demonstrated the use of a spinning bioreactor to provide improved nutrient and oxygen supply during organoid manufacturing (Lancaster et al., 2013). Using cellular

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memory devices that are a key development in the field of synthetic biology can help store all the

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transient responses of the organoids for a longer period (Purcell and Lu, 2014). Allenby et al. fabricated an efficient manufacturing tool based on ceramic hollow fiber constructs that when incorporated into 3D tissue models facilitate the separation of mononuclear cells( MNCs) and red blood cells ( RBCs) and improve tissue vasculature (Allenby et al., 2018). Merge of organoid technology & CRISPR Cas9 genome editing: Drost et al. demonstrated the deletion of key DNA repair genes of human colon organoids using CRISPR/Cas9 technology to study the origin of the characteristic mutations of cancer (Drost et al., 2017). Organoids have been developed from prostate cancer patients as in vitro prostate cancer models (Gao et al.). Attempts were made to generate cardiac organoids by combining CRISPR/Cas 9 genome editing tool and hiPSCs in order to correct the mutations that cause heart diseases and model heart diseases (Nugraha et al., 2019). To study the significance of

Journal Pre-proof hyperproliferation of colorectal cells and the pathogenesis of colorectal cancer, Crespo et al. demonstrated the idea of “disease in a dish” by generating colonic organoids using iPSCs derived from patients with familial adenomatous polyposis (Crespo et al., 2017). O’Rourke et al. succeeded in producing colorectal cancer models for future genetic and pre-clinical studies through the orthotopic engraftment of colon organoids (O'Rourke et al., 2017). The merge of organoid technology and CRISPR-Cas 9 genome editing has been a remarkable milestone in the field of cancer research as well as study of other disease and definitely adds value to research on organoids (Driehuis and Clevers, 2017; Lokody, 2013; Matano et al., 2015; Nie and Hashino,

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2017; Schwank et al., 2013; Wang et al., 2017) (Kim et al., 2019).

6. Conclusion

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In this review, we have deliberated on the main methodologies to fabricate organoids. Each of the methods has their own advantages and limitations. We have discussed some key protocols

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involved in the production of organoids and broadly classified them into four main categories (i)

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suspension culture system (ii) Systems using ECM (Matrigel) scaffolds, (iii) methods using spinning bioreactors and (iv) Air liquid interface (ALI) methods. ALI (3D culture system) can be

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integrated with various bioengineering technique such as bioprinting (Choudhury et al., 2018a) and microfabricated devices such as microfluidic organ-on-chip devices (Yu and Choudhury, 2019; Yu et al., 2019). Microfluidic organoid-on-a-chip platforms can ensure better nutrient and

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gaseous exchange enabling mimicking of 3D tissue architecture and physiology. Although to

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increase the critical size of the organoids, greater efforts are needed for ensuring organoids vascularization; either via generating artificial blood vessels or spontaneously generating vessels by stimulating the angiogenic pathways. A more interdisciplinary approach with efforts from both biologists and bioengineers may solve most of the current limitations, making way towards manufacturing of organoids (Huh et al., 2011; Kimura et al., 2018). In future, there is a possibility that all of the methods mentioned in the paper could be used interchangeably at various stages regardless of the source to obtain the optimal organoids. Meanwhile, a non-profit organization called HUB (Hubrecht Organoid Technology) has initiated and established “Living Biobank”. It was created with an aim to collect all well-characterized organoids generated from patient tissues. Such a centralized system of organoids “Living Biobank” will be beneficial for scientific research, personalized medicine, and drug

Journal Pre-proof development. At present, organoids produced are transported by immersing the organoids in liquid nitrogen (Xue and Shah, 2013), the organoids can be stored by freezing and thawed when used later, however, this affects the electrical activity inside the organoids (Chakradhar, 2016). Much work is also needed in finding a more efficient and sophisticated organoid transportation method for large-scale transportation. There are few important organoid companies that are looking into manufacturing and commercialing organoids in a large scale. Some of these includeCellesce, Organome, DefiniGEN, Prellis Biologics and Novoheart. Currently these organoids are purchased mostly by researchers hence the scale is still limited. Future holds the key to large

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scale commercial organoid production.

Acknowledgments

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We thank Dr Chen Sixun and Dr Wang Tianyi for insightful comments on the manuscript.

Funding

Author Contributions

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This work was supported by A*STAR JCO Grant 15302FG152 awarded to D.C.

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A.A.: manuscript writing including the figures and tables; F.Y.: manuscript writing including the figures and tables; D.C: conception and design, manuscript writing; W.H.: manuscript editing. A.A, D.C, F.Y. W.H: final approval of the manuscript.

Disclosure of potential conflict of interest The authors indicated no potential conflicts of interest.

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Journal Pre-proof Table 1. Summary of various organoids fabricated using different methods

Magneti c Levitati on method using BioAssemb ler plates

Lung Orga noid, Saliv ary Orga noid, adipo se tissue grow th

Organ oid forma tion by using only a scaffo ld such as Matri gel.

200– 250 μm diame ter (Saliv ary organ oids),

The respe ctive tissu e cells

―

―

Crypt Isolatio n, cell dissocia tion and cell culture techniq ue

Intest inal Orga noid

Adul t intest inal stem cells (Lgr 5+ are mark ers)

Produci ng organoi ds by

Intest inal Orga noid

huma n ESCs and

Demonst rated applicati on

Challen ges in using this method ology

―

―

Prompt cell-cell interacti ons and 3D structur es develop ment and accurate ly mirrors the in vivo tissue represe ntation

Current methodeasy and effective.

―

(Daquin ag et al.; Ferreira et al., 2019; Tseng et al., 2013)

Organoi ds were cultured along with their characte ristic features for more than 8 months

Facilitates further studies on gene therapy and regenerati ve medicine

Matrigel (a mouse sarcoma product) , not compati ble with human organ transpla ntations

(Sato et al., 2009)

The cells of the intestin

Facilitates molecular study on human

―

(Spence et al., 2011)

―

―

―

14 days

―

Goblet cells, Paneth cells, enteroen docrine cells and maturati on of brush border are observe d

―

―

Minim um 28 days impro

―

Presence of apical microvil li brush

Enables futuristic possibiliti es of studying inherited kidney diseases

Variabili ty of hPSCs cell lines affects different iation efficienc y and interstiti al space cells of kidney organoid s- not well defined

Referen ce

Each organoid produce d contains multiple nephron structure s

of

kidney organo ids (mass produc tion) within 12 days from NPCs

Advant age of this method

Allows examin ation of cells at each stage of differen tiation. The protoco l is effectiv e for both 2D and 3D organoi ds

ro

0.5 mm in diame ter

Three to six wells of a 24well plate are suffic ient to gener ate 96 organ oids in 3D cultur e.

Cost of the proc ess

-p

Size of the organ oid

Durati on to get the compl ete organ oid

re

hiPS Cs & hES Cs

Jo

3D Cultu re Syste m for Orga noids

Kidn ey Orga noid

Cell Sour ce

Num ber of orga noids

lP

Suspe nsion cultur e syste m for organ oids

96 well ultralow attachm ent plate method for suspens ion cultures

Type of Orga noid Prod uced

na

Meth ods to prod uce organ oids

Comple xity of organoi d – in terms of cells present in the organoi d formed etc.

ur

Orga noid cultu re syste m

Landm ark studies with slight variatio n in the protoco l

(Moriza ne and Bonvent re, 2017)

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The epitheliu m of hGOs exhibits surface mucous cells, contains antral gland cells and endocrin e cells

―

Duct tissu e or singl e cell

―

10-30 organ oids per well

Organ oid forma tion Via Embr yoid Form ation & Spinn ing

Cerebra l organoi d culture system

Retin al organ oid

huma n ESCs

Cere bral Orga noid

Mou se ESCs , ESCs or iPSC of huma ns.

Mainly used to create models to study disease caused by Helicobac tor pylori

―

Allows detailed study of epitheli almesenc hymal interacti ons

To study genetic modificati ons in organoids

Organoi ds produce d have large, continu ous epitheli al structur es

Phenotypi c and molecular heterogen eity build scope for organoido gensis and downstrea m applicatio n

study the cause of hypopla sia seen in microce phaly and can be maintai ned as

The organoid thus obtained can be used as a model to study neurodev elopment and neurologi

―

60 organ oids in total at the end of their study

21 days

―

Robust retinoge nesis is observe d with significa nt appeara nce of CRX cells & photorec eptors

Grow s up to 4mm in diame ter

Two 60m m plate conta ins 32 organ oids in total

8-10 days: basic neural identiti es appear . 20-30 days: define d brain region

$150 per orga noid not inclu ding the biore actor

Histolog ical analysis revealed regions of cerebral cortex, choroid plexus, retina and

ur

Jo

Retinal organoi ds produce d by neuroep ithelial Trisecti on method.

congenita l gut disorders

Ductal markers surround ing the central lumen resembli ng liver buds

re

Liver and Pancr eatic Orga noid

It takes about 1 to 4 weeks for organo ids to develo p.

al organoi ds acquire both secretiv e and absorpti ve functio ns Robust method to generat e hGOs to study stomac h develop ment and mechan isms involve d

of

―

This is a 34 days protoc ol to produc e human gastric organo ids

-p

―

border similar to the ones found in the mature intestine

na

Protoco l to produce both liver and pancrea s organoi d

Gastr ic Orga noid

huma n ESCs and hiPS Cs

ved develo pment observ ed in the organo ids

lP

Gastric Organoi d differen tiation protocol - from spheroi ds to organoi ds

hiPS Cs

ro

manipul ating a series of growth factors

―

Suitable only for adult epithelia l tissue, impedes exponen tial longterm expansio n of tissue in-vitro 1/3 of organoid s degrade d before day 21 and low OV formatio n frequenc y severely affects organoid number produce d. Tissue maturati on and neural & nonneural tissue crosstalks due to lack of surround

(McCra cken et al., 2014)

(Broutie r et al., 2016)

(Volkne r et al., 2016)

(Lancast er and Knoblic h, 2014; Lancast er et al., 2013)

Journal Pre-proof

huma n iPSC s

―

―

33 days

Large polarize d neuroepi thelial surround ing the fluid filled cavities with expressi on of CD133 at the apical surface resembli ng the ventricul ar zone

―

ro

Cere bral Orga noid

meninge s

re

-p

Organoi d produce d combini ng stem cell biology & Organ on chip technol ogy

s to form

―

31 huma n cereb ral organ oids

Organ oids are grown for 9 month s or more to establi sh mature brain feature s

Protoco l to produce mature cerebral organoi ds

Cere bral Orga noid

huma n PSCs

―

cal diseases

ing embryon ic tissue

The organoids thus obtained are a better model to study drug toxicity, brain developm ent and neurologi cal diseases

―

(Wang et al., 2018)

―

Organoi ds produce d exhibits a high number of SCone photorec eptors with typical morphol ogy along with a high expressi on of Soptin

Retinal organoi ds show improv ed maturat ion (Day25 organoi ds resembl ed Day 32 organoi ds of static culture system)

Use of bioreactor accelerate s the productio n, growth and differenti ation of organoids and allows retinal diseases and therapeuti c studies

Retinal organoid s produce d show signs of degener ation after day 25

(DiStefa no et al., 2018)

―

Mature structure s of brain that includes dendritic spinelike structure s are observe d

Maturat ion of organoi ds can be attained by light simulati on of photose nsitive cells

These mature brain organoids have the potential to model brain functions exhibited by adult brain

The role of light in modulati ng the neuronal network and photose nsitivity of cellsyet to be discover ed

(Quadra to et al., 2017)

lP ―

na

mous e PSCs

ur

Retin al organ oid

Appea rance of optic cup, increas e in growth till 25th day.

Jo

Retinal organoi ds producti on using rotating wall vessel bioreact or

such for over a year (around 15 months) Minima l cell death in the core of the brain organoi d observe d via TUNEL Assay indicati ng high viabilit y of brain organoi d on chip with improv ed maturat ion

of

Biore actor

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―

7-day monol ayer culture . 18 days 3 D culture to genera te kidney organo ids

―

Inducti on all the four types of progeni tors essentia l to generat e kidney organoi d that highly matches the first trimeste r human kidney.

Robust renal model to study human kidney developm ent, diseases & nephrotox ic drug screening and

Organoi ds remaine d healthy over a very long period (over 5 months)

Provides avenues to study developm ent and response to injury of human CNS

Organoi d culture conditio ns require further optimiza tion to reach Maturati on

(Takasat o et al., 2016)

―

(Giando menico et al., 2018)

―

-p

1-3 organ oids per mold

ur

―

re

huma n ESCs

lP

Cere bral Orga noid

Appro x 5060 and in some cases 90 days

ro

of

hPS Cs and hiPS Cs

na

Cerebra l organoi d generat ed using airliquid interfac e method

Kidn ey Orga noid

Jo

Organ oid produ ction using AirLiqui d interf ace Meth od

Differe ntiation Protoco l to produce kidney organoi d

6mm in diame ter at the end of 18th day of organ oid cultur e

Collecti ng ducts, loops of Henle, proxima l tubules, early mesangi al & distal tubules, glomeru li, tubular structure s and nephron s are observe d. Remark able selforganiza tion of callosal and corticof ugal tracts observe d

Journal Pre-proof Captions for Figures and Tables

Figure 1: Summary of the methods for fabrication of organoids Fig 2: Suspension culture system for fabrication of organoids Fig 3: Crypt isolation culture system: Isolation of crypt from primary intestine tissues Fig 3A: Crypt isolation culture system: Generating organoids from isolated crypts Fig 4: Cerebral organoid culture system. Fig 5: Air-liquid interface method for fabrication of organoids

Jo

ur

na

lP

re

-p

ro

of

Fig 6: Summary of factors crucial for the manufacturing of organoids

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6