Towards organoid culture without matrigel

ABSTRACT Organoids—cellular aggregates derived from stem or progenitor cells that recapitulate organ function in miniature—are of growing interest in developmental biology and medicine.

Organoids have been developed for organs and tissues such as the liver, gut, brain, and pancreas; they are used as organ surrogates to study a wide range of questions in basic and

developmental biology, genetic disorders, and therapies. However, many organoids reported to date have been cultured in Matrigel, which is prepared from the secretion of

Engelbreth-Holm-Swarm mouse sarcoma cells; Matrigel is complex and poorly defined. This complexity makes it difficult to elucidate Matrigel-specific factors governing organoid development.

In this review, we discuss promising Matrigel-free methods for the generation and maintenance of organoids that use decellularized extracellular matrix (ECM), synthetic hydrogels, or

gel-forming recombinant proteins. SIMILAR CONTENT BEING VIEWED BY OTHERS ORGANOIDS Article 01 December 2022 TISSUE EXTRACELLULAR MATRIX HYDROGELS AS ALTERNATIVES TO MATRIGEL FOR CULTURING

GASTROINTESTINAL ORGANOIDS Article Open access 30 March 2022 ENGINEERING ORGANOIDS Article 19 February 2021 INTRODUCTION Organoids are multicellular structures derived from stem and

progenitor cells that mimic the function and spatial organization of organs1. Organoids recapitulate important organ functions in vitro while remaining small in size and often free of

interfering cell types such as vascular, nerve, or other undesired epithelial cells. For these reasons, organoids are used to study organ development2 and model various diseases such as

cancers3, neural disorders4, and autism5; they are also used as pharmaceutical testing platforms6, model systems for CRISPR-CAS9-mediated treatment of genetic diseases7, and replacement

organs for transplantation8,9. It is possible to culture organoids from induced pluripotent stem cells (iPSCs)10 or adult stem cells from patient tissues11, which may lead to personalized

medicine. The wide range of clinical applications of organoids is the subject of a recent review by Drost and Clevers12. Many excellent reviews have been published about different organoid

types, such as heart13,14, brain15,16,17, liver18,19, kidney20,21,22, pancreas23,24,25, and female reproductive tract26. Many organoids have been cultured in Matrigel, a material derived

from the secretion of Engelbreth–Holm–Swarm mouse sarcoma cells and enriched for extracellular matrix (ECM) proteins27. In an early report of organoid culture, Sato and colleagues grew

murine intestinal Lgr5+ stem cells in high concentrations of Matrigel supplemented with the growth factors WNT, Noggin, R-spondin, and EGF28. This culture system has been widely adapted for

other organs such as the colon, stomach, and liver29,30,31,32,33. Related methods have been used to construct simulated versions of the inner ear34 and pancreas35,36,37,38,39, and the

similarities between pancreatic and liver-organoid-generating cells suggest that methods used for making liver organoids may be applicable to the pancreas40. The many organoid-specific

applications of Matrigel-based culture methods have been thoroughly discussed elsewhere41,42,43. Despite its versatility and affordability, Matrigel is extremely complex; proteomic analysis

shows that it contains more than 1800 unique proteins44. The undefined nature of Matrigel makes it difficult to identify the signals necessary for organoid structure and function; this

difficulty is compounded by lot-to-lot variations of Matrigel45,46,47,48. Furthermore, Matrigel may not contain all of the necessary components for proper organoid formation; gut organoids

cultured in Matrigel lack the characteristic architecture of mammalian intestines, which could be due to a sub-optimal amount of laminin-511 and the absence of other cell types such as

mesenchymal cells49,50. Finally, it has become increasingly clear that the mechanical properties in three-dimensional (3D) culture systems can have large effects on cell51, organoid52,

tissue53, and organ development54,55. The mechanical properties, such as elastic modulus, pore size, stress relaxation, and creep56,57,58,59, cannot be easily separated from the chemical

cues in the Matrigel-based culture systems. Furthermore, the mechanical properties of Matrigel samples are heterogeneous; local regions of such gels have been found to exhibit elastic moduli

several times higher than the average modulus of the sample60,61. Finally, the fact that Matrigel is originated from mouse cells hampers its use in human clinical transplantation due to

potential immunogenicity18. Given these limitations, there is an emerging need to develop Matrigel-independent organoid culture methods. In this review, we discuss recently-developed

Matrigel-free techniques for the culture of organoids. We will review undefined matrices, focusing on ECM derived from decellularized tissues and collagen, and defined matrices, including

synthetic polymer hydrogels and engineered ECM proteins (Fig. 1). Table 1 summarizes the advantages and disadvantages of each category of material. Table 2 summarizes studies that discuss

the effects of elastic modulus on the organoid formation of various tissues. ORGANOID CULTURE IN DECELLULARIZED ECM AND OTHER NATURALLY-DERIVED PROTEINS In organ development, ECM proteins

provide signaling cues, serve as an adhesive substrate, and sequester growth factors (Fig. 2)62. In order to accurately recapitulate the composition, structure, and vascularization of native

ECM in organ development, some organoids have been grown in decellularized ECM from human or animal donors. The methods of decellularization used are dependent on the target tissue and not

readily generalizable; a number of these methods have been reviewed elsewhere63. While xenogeneic ECM has the potential to cause immune responses, this risk can be greatly reduced by using

proper preparation techniques64; similar ECM scaffolds derived from animals are FDA-approved for clinical applications such as heart valve replacement, facial reconstruction, and osteopathic

implants62,65. Decellularized ECM may also provide additional cues that promote regeneration of damaged tissue, ultimately supporting the organoid transplant and promoting its function66.

Decellularization approaches have been demonstrated for human kidney67, murine kidney67,68, murine heart69,70, human and porcine lung71, and porcine testicular72 tissues, with each type

posing unique challenges. To illustrate some of these challenges and methods, we will focus on the decellularization of the liver, gut, and pancreas. LIVER ORGANOIDS GROWN IN DECELLULARIZED

ECM Liver-specific ECM can be obtained from a surgically resected portion of a patient’s damaged liver, or from livers unsuitable for transplantation. Lin and colleagues reported that liver

tissue decellularization supported growth and maintenance of rat hepatocytes; however, this method relied on mechanical disruption of resected tissue, which resulted in the loss of organ

architecture and vascular networks73. In contrast, Baptista and colleagues perfused Triton X-100 and ammonium hydroxide through a ferret hepatic vascular network to remove cells. This method

preserved the underlying ECM and vasculature while retaining most of the glycosaminoglycans, collagens, and elastins. The decellularized material could be colonized by human fetal liver and

endothelial cells to produce a functioning organoid74. An illustration of the method of Baptista and coworkers is shown in Fig. 3. The liver can also be decellularized, ground into powder,

and redissolved. Lee and colleagues used this approach with rat liver ECM to promote the differentiation of human adipose-derived stem cells into functional hepatocytes75. More recently,

Saheli and colleagues seeded sheep liver ECM gel with a combination of human hepatocarcinoma cells, mesenchymal stem cells, and umbilical cord stem cells; the resulting tumor organoids had

greater hepatocyte function than tumor organoids grown in comparable collagen I-based culture76. Lewis and colleagues observed that growing murine small cholangiocytes (a committed

progenitor cell type) in porcine liver ECM gel resulted in the formation of complex, branching structures similar to biliary ducts, and these cells also secreted small amounts of bile. In

contrast, cholangiocytes cultured in Matrigel formed cysts while those in collagen I proliferated and spread in all directions without spontaneously forming structures77. Thus, Matrigel

alone does not provide all of the needed factors for small cholangiocyte differentiation, whereas a decellularized liver ECM gel appears to be a better alternative. GUT ORGANOIDS GROWN IN

DECELLULARIZED ECM Decellularized matrices have also been used for the growth of human intestinal organoids (HIOs) derived from pluripotent stem cells (PSCs), as well as enteroids derived

from adult crypt stem cells78. Finkbeiner and colleagues found that undifferentiated human embryonic stem cells (ESCs) could not directly differentiate into HIOs in a decellularized porcine

intestinal ECM. However, pre-differentiated HIOs were able to seed onto decellularized porcine intestinal ECM and form correct spatial orientation-mimicking intestine79. Giobbe and

colleagues developed a method for decellularizing the porcine small intestine to form an intestinal ECM gel similar to the liver ECM gels discussed in the previous section. The porcine

intestinal ECM gel was able to support the formation of enteroids from murine Lgr5+ crypt cells, and from human pediatric stomach and intestinal crypts. The authors were also able to achieve

mechanical control of the ECM gel by incorporating poly-acrylamide to achieve different stiffness for two-dimensional (2D) culture of human and mouse enteroids, which may be important for

future research. This intestinal ECM gel is also applicable to grow organoids from the liver, stomach, and pancreas80. DECELLULARIZED ECM FROM THE PANCREAS Decellularized ECM has been

prepared from various pancreatic cell sources, such as adult human pancreas81 and porcine pancreas82. Using mass spectrometry, Bi and colleagues found major differences in protein

compositions comparing decellularized rat pancreatic extracellular matrix to Matrigel; Matrigel contains lower levels of collagen V than are normally present in pancreatic ECM. By coating

plates with Matrigel plus commercially available collagen V, the authors were able to enhance endocrine differentiation of human iPSCs in 2D culture, compared to Matrigel alone83. One key

limitation of this study, however, is that the proteomic profile of the rat pancreas may be very different from that of the human pancreas. The human pancreas presents a challenge in

decellularization as it has a higher lipid content than animal models. With specific preparation methods that remove lipids, Sackett et al. found that decellularized human ECM is capable of

supporting the survival of undifferentiated hPSCs and their pancreatic lineage derivatives, including insulin-expressing beta-like cells, in vitro81. TISSUE ORGANOIDS GROWN IN OTHER

NATURALLY-DERIVED PROTEINS AND BIOMACROMOLECULES A complementary approach to the use of decellularized ECM is naturally-derived proteins, such as collagen I derived from porcine tendon,

porcine skin, or bovine lens capsules. Collagen I has been used to form human colorectal carcinoma model organoids in a 3D culture of rabbit colons84 and as a support for the culture of

human and murine intestinal, stomach, and colonic organoids85. Other examples are vitrified collagen I for human intestinal organoids86 and murine renal organoids87, as well as fibrin

supplemented with laminin-111 capable of supporting various murine and human epithelial organoids88. The Tokyo Medical and Dental University (TMDU) method uses collagen I for intestinal

enteroid culture89. Yui and colleagues showed that embedding intact murine colonic crypts and isolated Lgr5+ progenitor cells in collagen I with hepatocyte growth factor, R-spondin 1, EGF,

and Noggin generated organoids that were able to engraft onto damaged mouse intestinal epithelia upon transplantation. In contrast to the TMDU method, the Ootani method uses collagen I gel

in which small and large intestinal cells are kept suspended at an air-liquid interface; this method improves oxygenation of the organoid, allows viable murine organoids to be maintained in

culture for up to 350 days, and preserves the mesenchymal niche in the organoids90. Two factors have been shown to affect the differentiation of progenitor cells into organoids in

collagen-based matrices: the source of seeded cells, and the spatial arrangement of collagen types around the cells. Isshiki and colleagues reported that the choice between the TMDU and

Ootani methods should be governed by the source of cells used to generate the intestinal organoid. More consistent results are obtained for growing intestinal organoids from seeded isolated

rat intestinal crypts using the TMDU method, whereas the Ootani method is better for growing rat colon organoid cultures of homogenized tissue91. In addition to judicious selection of cell

type, cell fate is intimately tied to interactions between cell surface integrins and biochemical cues in the ECM92. Collagen I has a high affinity for α2β1 integrin, whereas collagen IV

binds more strongly to α1β1 integrin93. Collagen IV tends to occur exclusively in basement membranes93. β4 integrin expressed by intestinal organoids is distributed only at the basal

surface94, while β1 integrin is required for proper apical-basal polarization95,96,97. A combination of collagen I and fibronectin compared to collagen I alone functionalized within a PEG

gel enhanced hepatic differentiation from human mesenchymal stem cells98. Clearly, the culture of organoids must take the 3D spatial positioning of the relevant materials into account. In

addition to organoids grown in naturally-derived proteins, several laboratories have grown a wide range of organoids in polysaccharides such as alginate or alginate-chitosan mixtures.

Organoid types grown in alginate include human lung99,100, human brain101, murine intestinal102, human intestinal102,103, human pancreatic104, and human and murine vascular105. Capeling and

colleagues grew HIOs on an alginate substrate and found that differentiation of human pluripotent cells into HIOs could be supported without the alginate providing chemical cues to the

cells. The authors hypothesized that cells create their own niche within the alginate hydrogel by secreting basement membrane proteins and forming mesenchyme, allowing cellular survival and

differentiation into HIOs103. Rossen and colleagues demonstrated the development of murine and human vascular organoids in a non-functionalized alginate setting105. Alginate has a number of

advantages that make it attractive as a material for further study; it is inexpensive, relatively easy to modify and functionalize, biocompatible, and has been used in a wide range of

biological and materials applications106,107,108. The mechanical properties of alginate, such as elastic modulus, extensibility, and characteristic relaxation time, can also be easily

tuned109. For these reasons, alginate is a promising material for further exploration. However, because alginate is biologically derived, its mechanical properties are still subject to

lot-to-lot variability110. Similarly, hyaluronic acid and mixtures of hyaluronic acid and chitosan have been extensively used in the growth and construction of neural

organoids111,112,113,114,115,116. ADVANTAGES AND DISADVANTAGES OF DECELLULARIZED ECM AND OTHER NATURALLY-DERIVED PROTEINS OR BIOMACROMOLECULES Decellularized ECM-based methods can quickly

recapitulate organ function. Many or all of the chemical cues required for the formation of a spatially-defined organ, including difficult-to-introduce glycoproteins, are already present,

minimizing the need for additional chemical modification of the ECM. Decellularized ECM retains the compositional differences observed between basal and apical regions. Collagen- and

alginate-based materials have been approved by the FDA for a wide variety of applications117, which allows for rapid clinical translation. Decellularized ECM does have disadvantages. Most

importantly, the quantity of ECM that is available for study is limited by the availability of donor animals or humans, and the quality of ECM can be affected by the health of a donor. For

example, emphysematous or fibrotic lung tissue has hardened and undergone alterations in its architecture. These alterations can lead to cells failing to survive beyond one week of

culture118 or broad changes in the phenotype of seeded cells that do survive119. Contrarily, myocardial infarct is known to trigger remodeling events that stiffen the ECM and change its

chemical composition; yet when mesenchymal stem cells are seeded on infarcted tissue, the cells secrete higher levels of pro-survival and immunomodulatory growth factors120. While myocardial

infarct appears to enhance the survival of seeded cells, the negative effects of other diseased tissue on organoid development should not be discounted. Even with healthy donor tissue,

batch-to-batch variability remains. The physical properties of decellularized ECM are difficult to control or modify, which limits the experiments that can be conducted. Decellularized ECM

is also chemically undefined; the factors driving differentiation are often unknown. Surface proteoglycans that are necessary for successful organoid formation may be removed by harsh

decellularization121. A related difficulty is that not all decellularization protocols are equally effective at removing cells or other immunogenic species, which can cause varying host

immune responses and failure of implants in clinical trials122. Finally, the occasional need for PSC differentiation into organ-specific progenitor cells that are then introduced into the

decellularized matrix requires an additional step. Collagen-based culture methods are not limited by donor tissue availability; biomedical-grade collagen can be harvested on an industrial

scale from cows and pigs. However, some collagen-based culture methods rely on coculture with supporting cells98, which introduces undefined components into the organoid culture.

Furthermore, it is difficult to modify the mechanical properties of these culture systems without altering chemical concentrations. To elucidate the effects of mechanical properties on

organoid development, researchers have turned to synthetic hydrogels that have been functionalized with cell-binding cues. ORGANOID CULTURE IN SYNTHETIC HYDROGELS Native ECM is complex; it

contains over 300 different proteins, each of which has a different biological function and stiffness123. This large number of proteins means that many variables cannot be easily dissected

to study the influences of ECM on organoid behavior and development. Synthetic hydrogels are attractive because their mechanical properties, functionality, and erosion rate can be

controlled. The matrix metalloprotease (MMP) family of enzymes affects cellular and organoid development by degrading ECM proteins124. By including MMP recognition sites on synthetic

hydrogels, it is possible to tune the rate of the hydrogel’s erosion. Manipulating synthetic hydrogels using methods such as electrospinning125, photopatterning126,127, spraying of

microspheres128, inkjet and 3D printing129, or microfluidic channels130 further enables control over the shape and size of the organoids. The ability to exert local control over chemical and

mechanical properties allows researchers to duplicate the heterogeneity in stiffness and composition found in organs, generate interfaces between materials similar to those found in the

ECM, and duplicate essential elements of material microstructure; each of these controls has implications for organ function and disease131,132. Synthetic hydrogels can also be made

responsive to external stimuli. For example, a thermoreversible hyaluronic acid- poly(_N_-isopropylacrylamide) (PNIPAAm) based hydrogel that solidifies at 37 oC and re-liquefies upon cooling

enabled culture and recovery of human pluripotent stem cells without enzymatically digesting the matrix133. Light-sensitive polyvinyl alcohol matrices have recently been developed for cell

culture; these matrices allow for control over the spacing of biochemical cues and the material environment134. Both of these materials may be useful for future organoid studies. The use of

synthetic hydrogels may also open up new avenues by altering the porosity of the scaffold on which the cells are grown. Dye and coworkers found that human lung organoids transplanted in mice

could merge together to form airway structures. However, this was only possible if the scaffold was able to degrade, which increased the material’s pore size135. Choi and coworkers reviewed

the pore sizes typically used in making tissue-engineering scaffolds, ranging from 5–15 microns for fibroblasts to 200–400 microns for osteoblasts, and developed a method for creating an

artificial kidney scaffold using microstereolithography136. However, these studies observed the effects of pore size on mature cells rather than organoid development from multipotent cells.

Broguiere and coworkers found that a Matrigel culture system had a pore size smaller than 200 nm, or the resolution limit of their confocal microscope, but that a fibrin-based material had a

pore size closer to 4 microns; these materials had comparable elastic moduli and colony-forming efficiency88. To our knowledge, studies showing an explicit connection between pore size and

organoid differentiation have not yet been reported. Synthetic hydrogels also have readily tunable viscoelastic properties, such as loss modulus and characteristic relaxation time. Many

relevant tissues are viscoelastic, with brain tissue in particular not only having a strong dissipative component (i.e., high loss modulus) in its response to stress137 but also having

slight differences in viscoelastic properties between white matter and gray matter138. The viscoelastic properties of a material affect matrix remodeling, cell spreading, migration,

differentiation, and consequently, organoid fate139,140,141. The effects of viscoelastic properties on cell culture and behavior are complex but have been thoroughly reviewed

elsewhere140,142. The effects of materials properties other than stiffness have only recently begun to be explored, and tunable hydrogels will enable more sophisticated experiments to be

conducted. THE ROLE OF CHEMICAL CUES IN ORGANOID DIFFERENTIATION Synthetic polymer-based culture allows organoid formation conditions to be evaluated using high-throughput

methods143,144,145. Synthetic hydrogels can be functionalized with biologically active moieties that permit the growth and spread of cells146,147,148,149,150,151; concentration and spacing

of these cues can be changed independently152,153,154,155. A striking demonstration of the utility of high-throughput approaches was provided by Ranga and colleagues, who prepared PEG-based

gels in 1536-well plates and studied murine ESCs (mESCs) expressing an Oct4-GFP reporter. The authors analyzed 1000 variations of matrix elastic modulus, cell-binding peptides, and matrix

susceptibility to MMP degradation and their effects on murine ESC fate156. The optimal conditions (elastic moduli ranged from 2–4 kPa and scaffolds with MMP insensitivity) produce murine

neural tube organoids that are more homogenous in colony size and morphology, as well as more polarized, than those grown in Matrigel. The percentage of cells containing an actomyosin

contractile ring is used as a metric for the polarity of the cells157. Cell-binding cues from collagen, fibronectin, or laminin have frequently been added to synthetic hydrogels to allow for

organoid growth and differentiation. Ng and colleagues created functional human liver organoids derived from human iPSCs in a colloidal crystal of PEG functionalized with collagen I,

fibronectin, or laminin-521158,159. Attachment of human iPSCs was successful in assemblies functionalized with collagen I and laminin-521 but not with fibronectin. This result builds on

previous work by the authors, in which basic human liver function was recapitulated by a PEG-based scaffold160. To promote iPSC differentiation into human neuronal progenitor cells, Ovadia

and colleagues compared photo-crosslinked PEG-based gels that contained chemical cues such as the laminin-derived cell-binding sequences YIGSR and IKVAV, the fibronectin-derived sequences

PHSRNG10RGDS and RGDS, and the vitronectin-derived sequence KKQRFRHRNRKG161. The authors found that PEG gels functionalized with YIGSR and PHSRNG10RGDS were permissive for human iPSC

survival and differentiation into neural progenitor cells in 3D culture. THE ROLE OF STIFFNESS IN ORGANOID DIFFERENTIATION The stiffness of synthetic hydrogels can be controlled and has an

effect on organoid formation. Gjorevski and colleagues reported a synthetic matrix for the intestinal organoid culture of murine and human Lgr5+ progenitor cells derived from the intestinal

crypt. The material consisted of a PEG gel functionalized with either an RGD fibronectin-derived peptide or a laminin-111-derived peptide162. A stiffer matrix containing an RGD

fibronectin-derived peptide promoted survival and proliferation of undifferentiated progenitor cells. In contrast, the softer matrix containing a laminin-111-derived peptide promoted the

differentiation of progenitor cells into functional murine and human organoids. The authors found that organoid formation in minimal nutrient conditions was permissible only within a narrow

range of matrix stiffness; the optimal elastic modulus was 190 Pa. Making the PEG gels more susceptible to MMP degradation resulted in depolarized organoids with irregular shapes. Major

findings of this study are illustrated in Fig. 4, which also demonstrates the effect of matrix stiffness and degradability on the formation of one class of organoids. Cruz-Acuna and

colleagues also found that PEG-based materials may need to be soft and degradable in order to support differentiated intestinal organoids. The authors used a 4-arm PEG maleimide to

encapsulate and culture Matrigel-derived HIOs. Organoid viability was reduced at high PEG density and matrix stiffness; RGD- or AG73 (CGGRKRLQVQLSIRT)-functionalized PEG matrices promoted

greater organoid viability than laminin-derived IKVAV- or type I collagen-derived GFOGER peptides163,164. After successful engraftment, HIOs generated from 4-arm PEG maleimide promoted the

healing of mucosal wounds in a mouse colon injury model. Intriguingly, when PEG was crosslinked with dithiothreitol to inhibit matrix degradation, organoid viability at seven days was poor

as measured by live-dead staining. This demonstrates a requirement of a degradable matrix for prolonged survival. SYNTHETIC HYDROGELS IN ACTION: MODELING DIFFICULT TISSUES SUCH AS THE BRAIN

AND PANCREAS Neural organoids present special challenges including a lack of reproducibility, batch-to-batch variation in transcriptional profiles, and susceptibility to small

microenvironmental changes that may have a considerable effect on organoid fate165. Essential elements of the complexity of the brain must be recapitulated to enable clinical applications of

neural organoids. For example, toxicity in brain tissue has many potential causes involving multiple cell types166,167,168. Therefore, the most useful organoids for toxicity models should

include multiple populations. Schwartz and colleagues used a PEG-based gel functionalized with pendant RGD cell-binding domains and crosslinked by MMP-degradable peptides to generate neural

organoids169. In this study, cells were introduced in three sequential stages: neural cells were introduced at day 0, vascular and mesenchymal stem cells at day 9, and microglia and

macrophage precursors at day 13. The organoids were then exposed to a library of known toxic and nontoxic compounds, and the resulting RNA-seq data of the organoid response were used to

build a machine-learning algorithm to assess the neurotoxicity of known and unknown compounds. In a blinded test, nine out of ten tested chemicals were correctly identified as toxic or

nontoxic; in contrast, the true positive rate of chemical identification in animal models is between 41 and 71%170. Brain organoids cultured in hyaluronic acid hydrogels were used to model

Down syndrome by Wu and colleagues114. The authors found that the differentiation of both normal and Down syndrome iPSCs into neurons was dependent on matrix stiffness; cells could be grown

at a softer elastic modulus of ca. 500 Pa but not ca. 1500 Pa, as indicated by higher expression of β-2 tubulin and microtubule-associated protein 2. However, Down syndrome patient-derived

iPSCs that were differentiated in the softer gel showed no discernable neurite outgrowth, suggesting a block in the maturation of the differentiated neurons. Hyaluronic acid hydrogels can

also be functionalized with various peptides to examine brain organoid differentiation. Lam and colleagues found that the concentration of laminin-derived IKVAV, with 300 μM being the

optimal concentration, was critical to neural organoid survival; however, this concentration did not enhance neuronal differentiation115. Bejoy and colleagues showed that functionalizing

hyaluronic acid with heparin affects neuronal patterning; the addition of heparin favored differentiation of human progenitors into neurons with a hindbrain fate, whereas non-functionalized

hyaluronic acid favored a forebrain fate. The authors also established that the stiffness of this hybrid material is relevant to cell fate determination; lower elastic moduli, ca. 300 Pa,

led to forebrain development, whereas higher elastic moduli, ca. 1000 Pa, led to hindbrain development116. Neural organoids have also been cultured in Matrigel-free conditions using

microfluidic approaches such as microwell printing. A number of groups were able to generate spheroids in chip-based devices171,172,173, but the spheroids lack spatial complexity and cell

types compared to fully-developed organoids. Without having to functionalize the well substrates, Chen and coworkers used a 3D printed mold to cast polydimethylsiloxane (PDMS) microwells to

generate human embryoid bodies that have the potential to differentiate into brain organoids in a suspension culture174. They found that a critical factor affecting differentiation was the

ridges of the culture vessel, unlike previous studies using smooth wells. This study represents a new direction towards the generation of organoids, where the shape of the culture vessel

might be tuned in order to change cellular phenotype, growth, and differentiation. Pancreatic organoids have proven difficult to prepare without resorting to Matrigel-based culture. To our

knowledge, Candiello and colleagues were the first group to use a synthetic hydrogel, amikacin hydrate crosslinked with poly(ethylene glycol) diglycidyl ether known as Amikagel, to culture

hESC-derived islet organoids175. Amikagels with elastic moduli ranging from 37 to 320 kPa were created, but no chemical signaling peptides were incorporated into the gels. The authors found

that stiffer gels drove pancreatic progenitor cells to aggregate, leading to increased differentiation and maturation into beta-like cells; this may have been mediated through paracrine

signaling enhanced by cellular proximity. Compared to cells grown in Matrigel, beta-like cells grown in Amikagel produced higher levels of functional beta-cell markers PDX1 and NKX6.1 and

were more responsive to a glucose challenge. Their finding challenges the conventional wisdom that cell-binding domains are required for effective organoid formation. The stiffness of the

gel is also very high, exceeding the elastic modulus of materials typically used in the culture of bone51. Mechanistically, the authors propose that the culture system is forcing the

formation of a compressed organ, rather than serving as a mimetic. A potential disadvantage of this approach is limited control of cellular aggregate sizes and attendant consequences of cell

viability of no more than five days. The smallest cell aggregates reported by the authors were ca. 200 microns in diameter; the typical human islet has a diameter of ca. 130 microns176.

Larger islets have been known to form necrotic centers because of a lack of oxygen and nutrient diffusion; other studies have found that islets best maintain cellular identity and function

when they have a size of ca. 100–150 microns177. Further work should be done in this system to establish long-term viability. ADVANTAGES AND DISADVANTAGES OF SYNTHETIC POLYMERIC MATRICES A

major advantage of using synthetic polymers for organoid culture is that they are amenable to systematic variation in structure and properties and can be used to explore the effects of

mechanical and chemical cues on cellular fate178,179. Moreover, many such materials, including PEG and PLGA, have been approved by the FDA for use in human therapeutics. Nguyen and

colleagues recently assessed more than 1200 synthetic polymer formulations for toxicity and abilities to promote implant vascularization and endothelial cell network formation180. This work

provides a valuable resource for organoid researchers, particularly those concerned with vascularization of organoids postimplantation. There are several disadvantages of synthetic

hydrogels. First, many synthetic hydrogels require the incorporation of biochemical cues such as cell-binding peptides. In the absence of biochemical cues cells may not attach to the

hydrogel, leading to anoikis (a type of programmed cell death)181 instead of organoid formation162. Improper spacing of biochemical cues can also lead to cell death182. While the backbone

materials of synthetic hydrogels are cheap and can be produced on an industrial scale, functionalization of these materials with precisely-placed, custom-made peptides significantly

increases cost and requires expertise in materials science, making these materials less attractive to cell biology labs. There have been a number of studies showing that organoids can be

grown on unmodified surfaces such as alginate, but this requires more research103,105,110,175,183. Further, synthetic hydrogels may degrade into cytotoxic by-products184 or require cytotoxic

initiators185, limiting the types of polymers that can be used in cell culture186. Synthetic hydrogels may contain pendant or other unreacted groups, which may be toxic to cells (such as

neurotoxic maleimides)187. Finally, synthetic hydrogels used as medical implants can trigger foreign body reactions188; similar effects may be seen in immunogen-containing organoids. For

these reasons, it may be advantageous to engineer recombinant protein gels. ORGANOID CULTURE IN PEPTIDE AND RECOMBINANT PROTEIN MATRICES Recombinant proteins made by genetically engineered

organisms have found wide applications in medicine, food processing, and catalysis. Engineered recombinant protein gels possess major advantages compared to other culture methods: chemical

cues can be added with exact definition; chemical and mechanical properties of the gel can be altered independently; polydispersity is low; and degradation rates can be programmed by

including appropriate recognition sites for MMP degradative enzymes. Chung and colleagues generated a hydrogel using an elastin-like polypeptide matrix containing a fibronectin-derived RGD

cell-binding domain with tetrakis(hydroxymethyl)phosphonium chloride (THPC) as an amine-reactive crosslinker; this material transiently inhibited contractility of murine ESC-derived

cardiomyocytes and enhanced survival of dorsal root ganglia cells from chick embryos189. In a follow-up study, murine cardiomyocyte differentiation (measured by α-myosin expression, cell

contractility, and metabolic activity) was found to be dependent on the stoichiometric ratio of THPC to protein, which tuned the stiffness of the hydrogel. Among the elastic moduli studied

(700, 3000, and 4000 Pa), the softest material favored the proliferation of embryoid bodies that contain mesodermal progenitor cells and promoted rapid cardiomyocyte differentiation (Fig. 

5). Embryoid bodies cultured in the 700 Pa matrix displayed the highest level of MMP secretion. Inhibition of MMP secretion was deleterious to proliferation and differentiation, suggesting

that remodeling of the matrix is essential in cardiomyocyte differentiation190. In another follow-up study, a very soft matrix with an elastic modulus of 180 Pa promoted intestinal

organoid-forming efficiencies comparable to those observed in collagen I-based matrices. Organoid-forming efficiency was higher when the engineered ECM proteins contained 3.2 mM RGD peptide,

compared to no RGD. Interestingly, MMP activity was significantly higher in the stiffer matrices. Inhibition of MMP activity reduced organoid-forming efficiency in the stiffer engineered

hydrogel matrices, suggesting that secretion of degradative enzymes in adult intestinal organoids may be a response to overly stiff conditions191. Recombinant ECM protein has been

investigated for pancreatic organoid culture. The Tirrell and Ku groups have jointly developed and studied an artificial elastin-like polypeptide that incorporates an 18-amino acid sequence

derived from α1 laminin; this polypeptide has been named artificial (a) ECM-lam (Fig. 6). aECM-lam was used to supplement a methylcellulose-based 3D pancreatic organoid culture that was

devoid of Matrigel. Adult murine Sox9/EGFP+ ductal progenitor cells were first proliferated in Matrigel, then transferred to a culture containing aECM-lam but not Matrigel. After 2 weeks,

endocrine-acinar organoids were observed, demonstrating that aECM-lam was capable of inducing differentiation of ductal progenitor cells into endocrine and acinar lineages192. A follow-up

study established that when Matrigel was added, endocrine and acinar cell development was inhibited while ductal cell formation was promoted193, demonstrating the importance of the ECM

microenvironment in pancreatic organoid differentiation. Using aECM-lam, other morphologically-distinct organoids were formed from murine postnatal pancreas194 and sorted adult ductal

progenitor cells195. Finally, the exact population of adult progenitor cells capable of giving rise to endocrine/acinar cells in aECM-lam was determined to be ductal cells, which have high

levels of CD133 but low levels of CD71 expression195. Collectively, these studies demonstrate the utility of aECM-lam in promoting endocrine and acinar cell differentiation in pancreatic

organoid culture and identifying the responsible progenitor population. Peptide-based hydrogels have recently been employed to model Alzheimer’s disease. Zhang and colleagues used the

self-assembling peptide RADA-16 to culture human neuronal cells treated with exogenous amyloid-β oligomers, known contributors to Alzheimer’s disease. A 3D culture in RADA-16 resulted in

activation of a p21-activated kinase in response to amyloid-β oligomers. Both the activation and localization patterns of the p21-activated kinase are characteristic of neurons in an

Alzheimer’s disease state. In contrast, the corresponding 2D culture did not show this activation and localization, suggesting that the 3D organoid culture of neurons is critical for

modeling Alzheimer’s disease196. The HYDROSAP self-assembling peptide hydrogel is a system recently developed by Pugliese, Marchini, and colleagues. In this system, multi-functionalized and

branched self-assembling peptides (SAPs) can generate hydrogels with controllable elastic moduli197. The authors used HYDROSAP peptide 3D hydrogels with elastic moduli of ~800 Pa (similar to

the stiffness of human brain tissue) to culture human fetal neural stem cells198, which were able to differentiate into various lineages including astrocytes, oligodendrocytes, and neurons.

Related work has been performed by Edelbrock and colleagues, with peptide amphiphiles capable of forming long, self-assembled nanostructures within a hydrogel. The peptides contain

brain-derived neurotrophic factor (BDNF), which enables the formation of mature neurons via activation of the TrkB pathway. Display of BDNF on the peptide amphiphile is necessary for this

effect to be observed199. Similar work has shown promise in stem cell differentiation and neural regeneration following spinal injury in vivo200,201,202, further demonstrating the utility of

peptide-based materials in cell culture. ADVANTAGES AND DISADVANTAGES OF RECOMBINANT PROTEIN MATRICES Self-assembling peptides and recombinant proteins offer important advantages in

organoid culture. Recombinant proteins are molecularly well-defined and can be tuned independently for stiffness, viscoelastic behavior, and chemical functionality203,204,205. They can be

programmed to degrade and remodel at controlled rates by including protease recognition sites206 or changing crosslinking chemistry207. Protein-based hydrogels can be outfitted with a broad

range of chemical functionalities by introducing noncanonical amino acids208,209; they can also be readily tailored to a wide variety of biomedical contexts210,211 and made thermally

responsive212,213. The programmability of recombinant proteins has prompted increasing interest in the design of protein-based hydrogels as matrices for organoid culture. Protein-based

materials have several disadvantages. First, not all proteins can be recombinantly expressed and ensuring re-folding and functionality of these proteins can be challenging. Certain

recombinant proteins and self-assembling peptides are immunogenic214,215,216,217,218. Ensuring that the recombinant protein is of human origin does not guarantee non-immunogenicity219. Care

must be taken to avoid introducing other immunogenic factors, such as bacterial endotoxin. Therefore, proteins for clinical use would preferably be expressed in mammalian expression systems

(e.g., Chinese Hamster Ovary) or in yeasts (e.g., _Pichia pastoris_). OUTLOOK AND CONCLUSIONS Although several Matrigel-free techniques have been developed, they have been used in a narrow

range of target tissues; expanding the number of tissue types will increase the acceptance of these alternative techniques. The ideal material for organoid culture should allow independent

changes in the chemical and mechanical properties so that the effects on organoid growth, development, or morphology can be correlated. It should also functionalize biologically-relevant

cell-binding proteins or peptides with ease. Finally, the ideal material should mimic the dynamic nature of the ECM in terms of erosion rate, viscoelasticity, and susceptibility to

degradation. Because of these requirements, synthetic materials and programmable recombinant proteins represent fruitful areas of future research. This review has proceeded with the

assumption that a matrix is required to culture organoids, but matrix-free culture systems have also been developed. For instance, Pagliuca and colleagues kept human embryonic stem cells

suspended in liquid culture at 70 rpm, added specific growth factors to encourage differentiation, and found that the resulting beta-like cells behaved similarly to mature beta cells220. A

similar process was used by Nair and colleagues using mechanical agitation to keep human cells suspended in culture, resulting in functional beta-like clusters221. Control over the

mechanical environment in such a system could be exerted by changing the speed of agitation. This method has the advantage of allowing easy harvesting of cells, which are simply allowed to

settle to the bottom of a tube. Using the methods discussed above, we anticipate a gradual shift away from the use of Matrigel in organoid culture and towards methods that enable exact

control of the cell’s mechanical and chemical environments with a more precise definition. REPORTING SUMMARY Further information on research design is available in the Nature Research

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thank Elena C. Chen for assisting in graphic illustration. We also thank Prof. David A. Tirrell for helpful discussions and editing assistance. M.T.K. was supported by the Department of

Defense through the National Defense Science & Engineering Graduate (NDSEG) Fellowship Program, and H.T.K. was supported by National Institutes of Health Grant R01DK099734. Support from

The Wanek Family Project for Type 1 Diabetes to H.T.K. is also gratefully acknowledged. The content is solely the responsibility of the authors and does not necessarily represent the

official views of the National Institutes of Health. AUTHOR INFORMATION Author notes * Christiana J. Crook Present address: Department of Medical Oncology and Therapeutics Research, City of

Hope National Medical Center, 1500 Duarte Rd., Duarte, CA, 91010, USA AUTHORS AND AFFILIATIONS * DEVCOM US Army Research Laboratory, Weapons and Materials Research Directorate, Science of

Extreme Materials Division, Polymers Branch, 6300 Rodman Rd. Building 4600, Aberdeen Proving Ground, Aberdeen, MD, 21005, USA Mark T. Kozlowski * Department of Translational Research and

Cellular Therapeutics, Diabetes and Metabolism Research Institute, City of Hope National Medical Center, 1500 Duarte Rd., Duarte, CA, 91010, USA Christiana J. Crook & Hsun Teresa Ku *

Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 1500 Duarte Rd., Duarte, CA, 91010, USA Christiana J. Crook & Hsun Teresa Ku Authors

* Mark T. Kozlowski View author publications You can also search for this author inPubMed Google Scholar * Christiana J. Crook View author publications You can also search for this author

inPubMed Google Scholar * Hsun Teresa Ku View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conception: M.T.K. and H.T.K.; Writing: M.T.K.,

H.T.K., and C.J.C. CORRESPONDING AUTHOR Correspondence to Mark T. Kozlowski. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW INFORMATION

_Communications Biology_ thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Anam Akhtar. Peer reviewer reports are available.

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