A Perspective on a Urine-Derived Kidney Tubuloid Biobank from Patients with Hereditary Tubulopathies

Abstract

Inherited kidney tubulopathies comprise a group of rare diseases with a significant societal impact, as lifelong treatment is often required and no therapies are available to prevent progression of renal damage. Diagnosis of inherited tubulopathies has improved with the advances of next generation sequencing. However, difficulties remain, such as a lack of genotype-phenotype correlation and unknown pathogenicity of newly identified variants. In addition, treatment remains mainly symptomatic. Both diagnosis and treatment can be improved by addition of in vitro functional studies to clinical care. Urine-derived kidney organoids (“tubuloids”) are a promising platform for these studies. International collections of patient-derived tubuloids in a living biobank offer additional advantages for drug development and pathophysiological studies. In this review, we discuss how diagnosis and treatment of tubulopathies can be improved by in vitro studies using a tubuloid biobank. We also address practical challenges in the development of such biobank.

Impact statement

This review provides readers insight into aspects related to diagnosis and treatment of hereditary kidney tubulopathies that can be improved. In addition, it explains why in vitro functional analyses using a kidney organoid model (tubuloids) may be useful as a method to improve these aspects. Finally, the additional advantages and practical hurdles of collecting tubuloid lines in a biobank are discussed.

Introduction

Inherited tubulopathies are a diverse group of rare diseases, involving every segment of the tubule, from the proximal tubule up to the collecting duct (for an overview, see Devuyst et al.¹). Genes that cause these diseases encode for transcription factors, as well as structural proteins, while genes for transporters and channels are predominantly affected. The societal impact of tubulopathies is high: it affects a patient's whole life, often presenting in childhood, leading to frequent hospitalization and sometimes to end-stage kidney disease. Because of the low incidence of individual inherited tubulopathies and potential misdiagnosis, epidemiological data have been difficult to establish.²

As a consequence of the low incidence of individual tubulopathies and the heterogenic clinical presentation, diagnosing and treating tubulopathies remain challenging. In this review, we propose kidney organoid cultures (“tubuloids”) as a platform for functional studies in tubulopathies that may improve diagnosis and therapy. Subsequently, we discuss the value of collections of tubuloid cultures in a biobank and address practical issues for its establishment.

Diagnostic challenges in genetic tubulopathies

The more widespread availability of genetic analyses, including next generation sequencing (NGS), has improved the diagnostics and our understanding of the molecular pathogenesis of hereditary tubulopathies.² However, NGS raised new issues, while other, already existing remain unresolved.

First, as a consequence of a more extended DNA analysis using NGS, new variants are identified on a regular basis. Interpreting these variants and predicting their pathogenicity are often difficult.³ The international guidelines on how to handle variants in established disease genes fall short in cases of new disease genes or complex inheritance patterns.⁴

Second, the relationship between mutation and disease phenotype is complex. This heterogeneity is strikingly illustrated in cases of Dent's disease, where the exact same mutation in one individual results in a more severe phenotype than in another individual, possibly as a result of differences in genetic background.⁵ In Gitelman syndrome, similar heterogeneity occurs, with variable severity of the disease phenotype among patients with the same mutation,⁶ including in individuals from the same family.⁷ Clearly, such heterogeneity makes it difficult to predict prognosis for an individual, and an incomplete disease phenotype may lead to misdiagnosis.⁸

Third, in a substantial number of patients with tubulopathy where a genetic cause is most likely, a genetic defect cannot be identified. In one-third of the pediatric patients with a high suspicion of a genetic tubulopathy, a genetic diagnosis cannot be established. For adult-onset disease, this holds for two-thirds of the cases.^9,10 In these studies, a limited number of genes were assessed, and more extensive DNA analyses (e.g., whole exome sequencing) are expected to increase the identification rate. However, due to unknown pathogenicity of variants, it is unlikely that in all cases disease-causing mutations will be identified using current approaches. Without known disease-causing mutations, family counseling is difficult, and the development and application of targeted therapies are complicated.

Therapeutic challenges in genetic tubulopathies

Treatment for hereditary tubulopathies is currently mainly symptomatic, lifelong, and does not prevent progression nor cure the disease. The rarity of individual disease, and even rarer specific mutations, complicates research into defect-directed (targeted) treatment strategies. Current strategies are often limited to correction of the clinical sequelae that result from the gene dysfunctions and, if required, combined with the generic treatment for chronic kidney disease. For channelopathies, lifelong treatment is required: Gitelman syndrome patients are treated with potassium suppletion, whereas nephrogenic diabetes insipidus patients require a sufficient fluid intake to balance water loss. For storage diseases, treatment is focused on prevention of accumulation of the detrimental metabolite: cysteamine reduces intracellular cystine content and is used as treatment for cystinosis. However, for the vast majority of the cystinosis patients, this only delays and does not prevent end-stage renal disease.¹¹ For cystic diseases, such as autosomal dominant polycystic kidney disease, there are few therapies that interfere with the primary process of cyst formation, and these approaches at best delay disease progression.

Fundamental studies to improve diagnostics and facilitate the development of new treatments

Functional in vitro studies on patient-derived cells can be used to provide insight into the pathophysiology of inherited tubulopathies, improve diagnosis, and contribute to the development of targeted treatments.

Regarding diagnostics, first, if cultured cells derived from patients with an inherited tubulopathy display an in vitro disease phenotype, functional studies can assess whether a newly observed or rare variant is pathogenic. Second, for diseases with an unpredictable genotype–phenotype relation, functional studies may aid in predicting a patient's ultimate disease outcome. Third, in cases where a genetic defect cannot be determined, cells from these patients may show an in vitro functional disease phenotype. This phenotype could yield clues toward the underlying genetic cause of the disease, for example, by comparing it to the phenotypes of a set of patients with known genetic defects (a similar phenotype may, in some cases, suggest a similar genetic defect).

Functional studies also hold promise for developing novel targeted treatment strategies. In vitro pathophysiological studies may identify therapeutic targets. High-throughput screening studies to assess the functional effects of a range of biologically active compounds and (repurposable) drugs may ultimately provide novel therapies.

Of note, increased understanding of the pathophysiology of inherited tubulopathies may lead to development of treatments for other more prevalent diseases: as analogy, the target for common antihypertensive drugs (loop diuretics) is the Na-K-2CL transporter, which is the substrate of Bartter syndrome.

Functional studies are also useful to predict treatment response in individual patients with known genetic defects and disease. It becomes increasingly more apparent that genetic testing is not sufficient to predict treatment efficacy of an individual patient. For some genetic diseases, functional assays have proven to be a better predictor. The most striking example is Cystic Fibrosis (CF): patients with exactly the same mutation in Cystic Fibrosis Transmembrane conductance Regulator (CFTR) may show different responses to treatment. For CF, functional organoid-based in vitro assays have been shown to accurately predict treatment efficacy,¹² illustrating the potential of functional assays for genetic disease.

For functional studies, human cells are preferred, as animal models do not always recapitulate disease phenotypes (reviewed by Iancu and Ashton⁴). Ideally, autologous cells are used, because genetic background contributes to disease phenotype, as illustrated for Gitelman, Dent's, as well as CF.^5,7,12 However, obtaining kidney tissue for the development and implementation of functional studies for hereditary tubulopathies is difficult, particularly since kidney biopsies are often not required for diagnosis. Each individual tubulopathy is rare, which makes the execution of clinical trials in these patients difficult or even impossible. Thus, the rarity and lack of tissue from patients with genetic tubular kidney disease hamper fundamental and clinical research in these patients.

Organoids as Platform for Functional Studies, Personalized Medicine, and Drug Development

Organoids are three-dimensional (3D) in vitro cultured epithelial structures that recapitulate key aspects of in vivo organs.¹³ These cultures can be established from pluripotent stem cells (PSCs; embryonic stem cells or induced pluripotent stem cells) or adult stem or progenitor cells (ASCs). For personalized medicine purposes, ASC-derived organoids^14–24 are generally more practical, as diseased tissue (e.g., tumor tissue) can directly be used to establish cultures, rather than going through a process of reprogramming and differentiation. The differences between ASC- and PSC-derived organoids, as well as the use of organoids for basic science and clinical applications, are more extensively reviewed elsewhere.^25,26 In this review, to illustrate the potential of ASC-derived organoid technology for personalized medicine and drug development, we highlight the example of CF—the disease for which clinical application of organoids has advanced furthest. Subsequently, we will discuss the potential of ASC-derived kidney tubular organoids (tubuloids) for inherited tubulopathies.

For CF, an organoid-based robust and high-throughput functional assay has been developed with straightforward readout to test treatment efficacy.²⁷ This forskolin-induced swelling assay in intestinal organoids is based on the fact that forskolin, through an increase in cyclic adenosine monophosphate (cAMP), opens CFTR. CFTR is located on the apical membrane in intestinal organoids, where it is the only cAMP-dependent channel. Upon opening, chloride moves from the cytoplasm into the lumen of the organoid, and water follows passively through osmosis, leading to swelling of the organoid. In CF patients, in which CFTR is not functional, the organoids do not swell. Thus, drugs can be tested for their ability to restore swelling and, hence, CFTR function. The response observed in the organoids correlates well with the patient's clinical response,^12,28,29 and in the Netherlands treatment decisions are made partially based on the outcome of this assay.

Recently, we described the novel methodology that allows the culture and expansion of human kidney tubular organoids (“tubuloids”) derived from kidney tissue and urine.^30,31 Establishing the cultures from tissue is highly efficient (close to 100%), whereas for urine efficacy currently reaches up to 20%. The tubuloids remain genetically stable over time; are expandable on the long term (approximately 15–20 passages); can be cryopreserved; and are relatively easy to handle with standard cell culture techniques. Tubuloids contain multiple epithelial cell types of distinct tubular segments (proximal, loop of Henle, distal, and collecting duct) that display functional transporter activity in culture. These characteristics render them useful for the modeling of physiology and disease.

Tubuloids can be cultured in standard well plates and transwells. In addition, to more closely model physiology, tubuloids can be cultured on an organ-on-a-chip plate, where the tubuloids form leak-tight tube-like structures.³⁰ On these plates, with distinct media inlets for the basal and apical side, the tubes display transepithelial transport function from the basal side to the apical side.

Tubuloids can also be used for disease modeling: when modeling infectious (BK-virus nephropathy) and oncological (Wilms tumor) disease, the tubuloids captured genetic and histological features of the in vivo disease.

Importantly, kidney tubuloid cultures can be established from urine from patients with genetic disease. As proof of principle, a tubuloid culture was developed from urine from a CF patient. CF was selected as disease, because a validated organoid-based functional assay (forskolin-induced swelling assay) to evaluate treatment efficacy was already established.²⁷ The forskolin-induced swelling assay in urine-derived tubuloids yielded comparable results to the intestinal organoids of the same patient that were tested in parallel. This indicates that urine-derived tubuloids also predict the patient response to therapy. Together, these findings show the potential of urine-derived kidney tubuloids as personalized genetic disease models. Interestingly, as urine-derived tubuloid cultures can be relatively easily (noninvasively) obtained, these cultures may serve as cell source for genetic diseases affecting other organs for which biopsies are cumbersome to take—as long as the substrate is expressed in kidney organoids.

Tubuloids also hold promise for inherited tubulopathies, because cells or genes involved in tubulopathies are present or expressed in tubuloids: proximal tubule cells are present, allowing the modeling of, for example, cystinosis and disease-causing genes, such as SLC12A1 (causing Bartter syndrome) that are expressed in culture.

Tubuloid Biobank for Hereditary Tubulopathies

Well-characterized sets of organoid lines derived from patients with similar diseases can be stored in organoid biobanks. Biobanks have already been developed and successfully used for pathophysiological research, as well as for personalized medicine and drug development.^20,23,32,33 Although biobanks have mainly been oncological in nature, the proof of principle for genetic disease biobanks was demonstrated by the establishment of a CF biobank, in which intestinal organoids from nearly half of the Dutch CF population (664 patients) were collected.³⁴

Organoid biobanks typically include a centrally stored collection of lines that can be made available to the scientific community. This requires well-organized informed consent in combination with privacy protection and tracking of samples. The latter factors are important to ensure that the organoids are used according to the informed consent, as well as to guarantee complete destruction of a specific organoid/tubuloid line in case of withdrawal of consent.

A centralized international biobank from patients with inherited tubulopathies would comprise 3D tubuloid cultures, derived from urine that can be cryopreserved. Such biobank will provide great possibilities to elucidate pathophysiological mechanisms and facilitate the investigation of larger cohorts of patients with “rare” diseases. The noninvasive nature of tubuloid generation from urine makes it patient friendly to participate. The expansion potential of tubuloids combined with cryopreservation enables shipment of samples to a centralized institute. Centralization will limit technical variation in the expansion and storage of organoids, as well as the execution of the experiments.

The tubuloid biobank can be of use in the evaluation of genetic variants of unknown pathogenicity. Pathogenicity of genetic variants may be assessed by comparing biobanked tubuloids of patients with a clear pathogenic variant, with those of an individual with an uncertain pathogenic variant. These comparisons may involve analyses on multiple levels, including RNA, protein, function, and morphology.

For diseases without a clear genotype/phenotype correlation, the functional tubuloid phenotype of a particular patient, relative to set of biobanked tubuloid samples of other patients with the same disease, may be of prognostic value. In addition, follow-up studies may shed light on the underlying causes of genotype–phenotype variability, by modification of the experimental conditions: for example, by inhibiting involved signaling pathways or modification of genes associated with the disease, using CRISPR-Cas9. This might provide insight in how genetic background modifies the pathogenicity of a variant.

A centralized biobank of tubuloids derived from patients with inherited tubulopathies will facilitate drug development in different ways. The biobank will enable testing of potential therapeutic compounds on specific subsets of patients. These subsets may comprise patients with a specific clinical disease (e.g., Bartter) or may concern patients with a similar type of defect (e.g., a premature stop codon in case of read through drugs or unstable or misfolded proteins in case of chaperone drugs) or subgroups in which the same signaling pathways are affected.

Once new and targeted treatments have been developed for hereditary tubulopathies, personalized in vitro drug screening to test efficacy of the treatment is likely preferred over treating all patients with that specific disease. Biobanked samples may be used to predict patient-specific efficacy of the new drug. Moreover, new treatments are typically patented and therefore expensive. This makes it preferable from an economic public health point of view to first test whether an individual is sensitive to an expensive treatment and only clinically treat those that are likely to benefit. In addition, this screening will limit ineffective treatment while having the patient unnecessarily exposed to the risk of developing adverse effects.

Challenges in establishing a biobank for hereditary tubulopathies

Although the potential of establishing a biobank of tubulopathies is evident, several challenges remain regarding the tubuloid model, the organization of the biobank, and the ethical and regulatory issues.

First, a few aspects of the tubuloid model would benefit from further development. Despite the high proliferative capacity, expansion of the cultures is limited to ∼15 passages. Although this yields sufficient material for drug screening experiments and characterization, increased expansion capacity would be preferable, particularly given the rarity of the diseases. It is expected that expansion capacity can be increased by adaptation of the culture media, as was done for other organoid cultures.³⁵ Also, establishing cultures from normal kidney tissue has nearly 100% success rate, but for urine, the process is less efficient (10–20%).³⁰ This could be improved by optimization of antibiotic regimens to reduce contaminations and by minimizing the time between voiding and processing the sample in the laboratory to decrease the time the cells are in a hostile environment. In addition, the tubular segment composition of tubuloid cultures differs from one culture to another: one culture may be more proximal tubule in nature, whereas another is more distal. For adequate disease modeling, the cellular composition should be determined for each tubuloid line, using for example segment-specific immunostainings or (single cell) RNA sequencing. Alternatively, after establishing a tubuloid line, the desired cell types or tubular segment for disease modeling can be fluorescence-activated cell-sorted.

Second, the organization and logistics of a biobank are complex and add new logistical challenges to outpatient clinics. Other organoid biobanks^20,23,33,34 illustrate the feasibility of implementing such logistics. In Box 1, we elaborate on specific logistic aspects, and a workflow is suggested (Fig. 1).

FIG. 1.

The logistics of the proposed tubuloid biobank, as described in Box 1. The first column (“when”) indicates when an action takes place: either before the patient's consultation, on the day of consultation, or after consultation. The second column (“where”) indicates where an action takes place: either at home (for the patient), the hospital, the research institute, or at the biobank storage. The third column (“what”) indicates what is done: the research nurse contacts the patient to ask whether a patient is interested to participate, and, if so, information and informed consent forms are sent to the patient; during consultation the informed consent forms are signed and safely stored, whereas the urine sample is pseudonymized by the research nurse and subsequently handed over to the researcher; the researcher establishes tubuloid cultures and cryopreserves a stock and hands these over to the biobank storage; at the biobank storage, the research manager handles requests for tubuloids by the scientific community and organizes potential withdrawals of consent. Color images are available online.

Box 1.

Logistics of the Tubuloid Biobank (with Fig. 1)

Here, we chronologically address organizational procedures involving biobanking, from patient inclusion until withdrawal, based on previously established biobanks.^20,23,33,34

For inclusion of patients, two variants are applicable, for patients that are known to the clinic and for patients that are new to the clinic. For known patients, a research nurse or the treating physician approaches the patient for consent ∼14 days before scheduled routine check-up. If the patient is interested, forms for consent are sent to the patient, at least a week before the scheduled appointment (in the Netherlands, a consideration period for study participation is a legal requirement). Patients who are interested in participation bring the forms to scheduled appointment to sign during the consultation. For new patients, the study is addressed by the treating physician in the first appointment. If interested, the informed consent process will be started afterward, and the forms are brought to the next check-up to sign.

The patients that are willing to participate (both new and known patients) receive a flask to collect urine after completing the consultation. Subsequently, the sample is, together with the informed consent forms, handed to the research nurse. The research nurse pseudonymizes the sample, to ensure that the researchers cannot track the identity of the patient. A researcher is only provided with the patient's gender, age, disease (phenotype), and, if known, genetic aberration.

Next, the researcher receives the pseudonymized urine sample (as soon as possible, to minimize delay in processing the sample). The research nurse stores the informed consent forms together with the pseudonymization information to decode a sample, in a dedicated and lockable office space.

If the researcher establishes cultures, frozen stocks will be stored centrally. A dedicated contact person, affiliated to the biobank and storage organization, rather than to the research group, will organize tubuloid lines and keep track of where samples have been shipped. This person—a dedicated contact person for both patient and researcher—also takes care of withdrawals of consent. To facilitate withdrawal of consent, even years after donation, contact information is provided to the patients and is available online.

As genetic tubular diseases are rare, (inter)national collaborations will be important to increase the sample size. Clearly, in case of international collaboration, distinct regulations and legal requirements from the countries involved should be aligned and adhered to.

Third, long-term storage of tubuloids of patients with inherited tubulopathies requires legal and ethical consideration. For optimal use of tubuloids, the cultures have to be combined with patient data: to be able to apply the tubuloids for personalized medicine, the cultures must be traceable to the patients. Tubuloid lines are therefore pseudonymized and not anonymized. Hence, organization of patient consents, also with regard to ownership and withdrawal of consent, is highly relevant. Organoid biobanks are useful for a broad area of research, across multiple fields and involving many scientists. Therefore, Bredenoord and colleagues^36–38 have proposed a broad consent, rather than the most common form of consent that is aimed at a specific research goal. In addition, regarding ownership, complex issues will occur, if for example a patentable compound is developed using urine-derived organoid cultures from a donor.^36,39 Which parties (the pharmaceutical company, the researcher, the biobank, the tissue donor) in such cases benefit could be addressed in the research proposal for the biobank that is reviewed by the medical-ethics committee.

Conclusion

A urine-derived organoid biobank of genetic kidney tubulopathies will be highly valuable. When logistics and ethics have been properly arranged, it will make kidney tissue available to the research community worldwide from a diverse group of patients with rare kidney diseases. Fundamental and functional studies will improve our understanding of pathophysiology; help to determine pathogenicity of new variants; and provide more insight into genotype–phenotype relationships. In addition, drug sensitivity may be tested on an individual level, whereas a biobank will allow high-throughput screening of potentially therapeutic compounds. In short, a biobank will facilitate the diagnostic process and the development of new treatments, ultimately improving patient care.

Footnotes

Acknowledgments

The authors thank Else Driehuis for the design of . The authors thank Hans Clevers for valuable discussions on the concept of organoid biobanking.

Disclosure Statement

All the authors have nothing to disclose.

Funding Information

This work was supported by the partners of Regenerative Medicine Crossing Borders, powered by Health∼Holland, Top Sector Life Sciences & Health. We gratefully acknowledge the Gravitation Program “Materials Driven Regeneration,” funded by the Netherlands Organization for Scientific Research (024.003.013).

References

Devuyst

, Knoers

N.V.A

.M., Remuzzi

, and Schaefer

Rare inherited kidney diseases: challenges, opportunities, and perspectives. Lancet, 383, 1844, 2014.

Mejía

, Santos

, Claverie-Martín

, et al. RenalTube: a network tool for clinical and genetic diagnosis of primary tubulopathies. Eur J Pediatr, 172, 775, 2013.

Richards

, Aziz

, Bale

, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, 17, 405, 2015.

Iancu

, and Ashton

Inherited renal tubulopathies—challenges and controversies. Genes, 11, 277, 2020.

Zhang

, Wang

, Xiao

, and Yao

Dent disease: same CLCN5 mutation but different phenotypes in two brothers in China. Intractable Rare Dis Res, 6, 114, 2017.

Coto

, Rodriguez

, Jeck

, et al. A new mutation (intron 9 + 1 G> T) in the SLC12A3 gene is linked to Gitelman syndrome in Gypsies. Kidney Int, 65, 25, 2004.

Lin

S.-H.

, Cheng

N.-L.

, Hsu

Y.-J.

, and Halperin

M.L.

Intrafamilial phenotype variability in patients with Gitelman syndrome having the same mutations in their thiazide-sensitive sodium/chloride cotransporter. Am J Kidney Dis, 43, 304, 2004.

Gianesello

, Del Prete

, Anglani

, and Calò

L.A.

Genetics and phenotypic heterogeneity of Dent disease: the dark side of the moon. Hum Genet, 140, 401, 2020.

Ashton

E.J.

, Legrand

, Benoit

, et al. Simultaneous sequencing of 37 genes identified causative mutations in the majority of children with renal tubulopathies. Kidney Int, 93, 961, 2018.

10.

Hureaux

, Ashton

, Dahan

, et al. High-throughput sequencing contributes to the diagnosis of tubulopathies and familial hypercalcemia hypocalciuria in adults. Kidney Int, 96, 1408, 2019.

11.

Brodin-Sartorius

, Tête

M.-J.

, Niaudet

, et al. Cysteamine therapy delays the progression of nephropathic cystinosis in late adolescents and adults. Kidney Int, 81, 179, 2012.

12.

Dekkers

J.F.

, Berkers

, Kruisselbrink

, et al. Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis. Sci Transl Med, 8, 344ra384, 2016.

13.

Clevers

Modeling development and disease with organoids. Cell, 165, 1586, 2016.

14.

Driehuis

, Kolders

, Spelier

, et al. Oral mucosal organoids as a potential platform for personalized cancer therapy. Cancer Discov, 9, 852, 2019.

15.

Sato

, Stange

D.E.

, Ferrante

, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology, 141, 1762, 2011.

16.

Bartfeld

, Bayram

, van de Wetering

, et al. In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection. Gastroenterology, 148, 126.e6, 2015.

17.

Huch

, Gehart

, van Boxtel

, et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell, 160, 299, 2015.

18.

Boj

S.F.

, Hwang

C.-I.

, Baker

L.A.

, et al. Organoid models of human and mouse ductal pancreatic cancer. Cell, 160, 324, 2015.

19.

, Gehart

, Artegiani

, et al. Long-term expansion of functional mouse and human hepatocytes as 3D organoids. Cell, 175, 1591, 2018.

20.

Sachs

, de Ligt

, Kopper

, et al. A living biobank of breast cancer organoids captures disease heterogeneity. Cell, 172, 373.e10, 2018.

21.

Sachs

, Papaspyropoulos

, Zomer-van Ommen

D.D.

, et al. Long-term expanding human airway organoids for disease modelling. EMBO J, 38, e100300, 2019.

22.

Karthaus

W.R.

, Iaquinta

P.J.

, Drost

, et al. Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell, 159, 163, 2014.

23.

Kopper

, de Witte

C.J.

, Lõhmussaar

, et al. An organoid platform for ovarian cancer captures intra-and interpatient heterogeneity. Nat Med, 25, 838, 2019.

24.

Lõhmussaar

, Kopper

, Korving

, et al. Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids. Nat Commun, 11, 1, 2020.

25.

Schutgens

, and Clevers

Human organoids: tools for understanding biology and treating diseases. Annu Rev Pathol, 15, 211, 2020.

26.

Drost

, and Clevers

Organoids in cancer research. Nat Rev Cancer, 18, 407, 2018.

27.

Dekkers

J.F.

, Wiegerinck

C.L.

, de Jonge

H.R.

, et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med, 19, 939, 2013.

28.

Berkers

, van Mourik

, Vonk

A.M.

, et al. Rectal organoids enable personalized treatment of cystic fibrosis. Cell Rep, 26, 1701.e3, 2019.

29.

de Winter-de Groot

K.M.

, Janssens

H.M.

, van Uum

R.T.

, et al. Stratifying infants with cystic fibrosis for disease severity using intestinal organoid swelling as a biomarker of CFTR function. Eur Respir J, 52, 1702529, 2018.

30.

Schutgens

, Rookmaaker

M.B.

, Margaritis

, et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Nat Biotechnol, 37, 303, 2019.

31.

Calandrini

, Schutgens

, Oka

, et al. An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity. Nat Commun, 11, 1, 2020.

32.

Broutier

, Mastrogiovanni

, Verstegen

M.M.

, et al. Human primary liver cancer–derived organoid cultures for disease modeling and drug screening. Nat Med, 23, 1424, 2017.

33.

van de Wetering

, Francies

H.E.

, Francis

J.M.

, et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell, 161, 933, 2015.

34.

Geurts

M.H.

, de Poel

, Amatngalim

G.D.

, et al. CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank. Cell Stem Cell, 26, 503.e7, 2020.

35.

Huch

, Gehart

, van Boxtel

, et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell, 160, 299, 2015.

36.

Bredenoord

A.L.

, Clevers

, and Knoblich

J.A.

Human tissues in a dish: the research and ethical implications of organoid technology. Science, 355, eaaf9414, 2017.

37.

Boers

S.N.

, Van Delden

J.J.M.

, and Bredenoord

A.L.

Broad consent is consent for governance. Am J Bioeth, 15, 53, 2015.

38.

Lensink

M.A.

, Jongsma

K.R.

, Boers

S.N.

, Noordhoek

J.J.

, Beekman

J.M.

, and Bredenoord

A.L.

Responsible use of organoids in precision medicine: the need for active participant involvement. Development, 147, 1, 2020.

39.

Boers

S.N.

, Van Delden

J.J.M.

, and Bredenoord

A.L.

Organoids as hybrids: ethical implications for the exchange of human tissues. J Med Ethics, 45, 131, 2019.