Extracellular matrix extract and hydrogel derivatives and uses thereof in cell and organoid culture

Abstract

The invention relates to methods for preparing an extracellular matrix (ECM) extract, hydrogel or derivative thereof, comprising providing organ tissue of an animal of the genus Bos, decellularizing the organ tissue, and digestion of the decellularized organ tissue. The invention further relates to EMC extracts, hydrogels and derivatives thereof, as well as to uses of such EMC extracts, hydrogels and derivatives thereof.

Description

[0001] P138583PC00

[0002] Title: Extracellular matrix extract and hydrogel derivatives and uses thereof in cell and organoid culture

[0003] Field of the invention

[0004] The invention relates to the field of extracellular matrix based extracts and hydrogels, in particular as a substrate for cell, tissue and organoid culture.

[0005] Background of the invention

[0006] In vitro cell culture work has proven to be highly effective for understanding both the fundamental principles governing physiology as well as pathophysiology and for identifying and even producing therapeutic and diagnostic molecules.

[0007] Further progress, however, is hampered by the fact that cell lines can substantially differ from their counterparts in vivo, because of the peculiar genetic abnormalities needed for a cell to thrive in the conditions of in vitro cell culture. This results in a number of experimental and functional limitations, including a relatively small repertoire of permanent cell lines available (e.g. the cancer cell line encyclopaedia contains only 1072 cell lines, less than the number of cancer types recognized by the pathologist), poor representation of the cellular physiology / pathophysiology respective to the source tissue from which the cell lines were generated, crippling safety concerns with respect to regenerative medicine, highly restricted availability of compatible immunological tools and very limited translational capacity in the development of novel therapeutics.

[0008] Many of these concerns can be addressed by using an alternative technology, involving the creation and culture of organoids, stem cell-derived 3D culture systems that re-create the architecture and physiology of organs in remarkable detail, while being fully amendable to unlimited expansion, genetic manipulation and immunological probing. Organoids are easily generated from almost any tissue type, even when only a minuscule amount of tissue is available, among other things allowing precision medicine. Thus organoid culture has now constitutes a sizeable part of all human in vitro experimentation and will replace the use of immortalized cancerous cell lines that has been used as flawed 2D culture models of human cells in the past 75 years. An important consideration is that organoid work critically relies on Matrigel®, a commercial product. Matrigel® is the solubilized basement membrane matrix secreted by the parietal endoderm-derived Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences. Matrigel resembles the laminin / collagen I V-rich basement membrane extracellular environment found in many tissues and is used as a substrate (basement membrane matrix) for culturing cells. However, Matrigel is produced by inoculating mice with the sarcoma cell line, an expensive, ethically-unacceptableand environmentally unfriendly process, plagued by batch-to-batch variation and safety-concerns.

[0009] Hence, there is still a need for an alternative hydrogel which overcomes one or more of the drawbacks indicated herein above.

[0010] Summary of the invention

[0011] It is an object of the present invention to provide extracellular matrix extracts, hydrogels and derivatives thereof, as well as method for preparing such ECM extracts, hydrogels and derivatives and uses thereof.

[0012] The invention therefore provides a method for preparing an extracellular matrix (ECM) extract, comprising:

[0013] - providing organ tissue of an animal of the genus Bos,

[0014] - decellularizing the organ tissue, and

[0015] - digestion, preferably enzymatic and / or chemical digestion, of the decellularized organ tissue.

[0016] In a further aspect, the invention provides an extracellular matrix (ECM) extract, hydrogel or derivative thereof obtainable by a method according to the invention.

[0017] In a further aspect, the invention provides an extracellular matrix (ECM) extract, hydrogel or derivative thereof, in particular derived from organ tissue of an animal of the genus Bos, comprising yak-derived collagen, yak-derived laminin, and / or yak-derived nidogen-1 and optionally other factors.

[0018] In a further aspect, the invention provides a use of an ECM extract, hydrogel or derivative thereof according to the invention as a substrate for cell, tissue or organoid culturing.

[0019] In a further aspect, the invention provides a cell, tissue or organoid culture substrate comprising an ECM extract, hydrogel or derivative thereof according to the invention.

[0020] In a further aspect, the invention provides a method for culturing cells comprising providing an ECM extract, hydrogel or derivative thereof according to the invention and culturing cells, preferably wherein the cells form a multicellular tissue or organoid.

[0021] Detailed description

[0022] As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of’ meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specificallyidentified, said additional component(s) not altering the unique characteristic of the invention.

[0023] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0024] The word “approximately” or “about” when used in association with a numerical value (e.g. approximately 10, about 10) preferably means that the value may be the given value (e.g. 10), plus or minus 5% of the value (e.g. 10, plus or minus 5%), preferably plus or minus 1% of the value.

[0025] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0026] The present inventors developed a extracellular matrix (ECM) extract based hydrogel from still-born foetal material derived from spontaneous crosses of yaks (Bos grunniens or Bos mutus) and dairy cows (Bos taurus or Bos indicus).

[0027] It was found that a hydrogel based on Yak extracellular matrix, derived from e.g. liver tissue, performs even better in sustaining living organoid culture as compared to Matrigel®, and the capacity of such matrix or tissue -specific matrix derived from yak calves for specific types of organs is a promising line for future research. The most important hallmark of organoids is their capacity to selforganize into three-dimensional structures. As demonstrated in the Examples herein, yak / cow derived hydrogel differs in several aspects from Matrigel® and BME, such as an increased collagen content and the presence of yak-derived and cow-derived peptide / protein. Furthermore, the yak / cow derived hydrogel allows for cells from liver biopsies to self-organize and differentiate into different structures, in particular polarised cholangiocyte-like epithelium with duct-like (cholangiocyte-like) and parenchyma-like (hepatocyte -like) compartments within the same culture. This differentiation occurred spontaneous without the need for expensive and complex differentiation procedures. Organoids grown form branching phenotypes, demonstrating a regenerative / growing phenotype of the biliary tract. Moreover, the presence of highly polarized cholangiocyte-like columnar epithelial layers was demonstrated. As such, it was shown that a hydrogel of the invention outperforms benchmark product Matrigel / BME as it allows for the development of more complex structures without the need for differentiation media. Gene expression profiling of intrahepatic cholangiocyte organoids (ICO) revealed that ICO grown in yak / cow derived hydrogel reflect a more mature cholangiocyte-like phenotype. Further important technical advantages include the scalability of the product. It is possible to create matrices of > 0.1 m3allowing applications as artificial full organ growth that are exceedingly challenging with current Matrigeland Matrigel-related product. Moreover, the substantially reduced batch to batch variability by pooling very large numbers (>1000) of foetal livers into a single batch of matrix. As demonstrated in the Examples herein, peptide correlations between different batches of a hydrogel of the invention range from 0.85 to 0.97, and collagen-peptide correlations range from 0.98 to 0.99, demonstrating high compositional similarity.

[0028] The societal advantages for such yak derived organoid-culture supporting matrices would be huge. Currently yields of Matrigel® production using the EHC mouse sarcoma cell inoculation technique are small and cumbersome: tumors are propagated by inoculating animals by trained zootechnical staff with 1 gram of minced tumor, which is grown to a mass of 4 / 5 grams, a process which approximately takes a month, while the animal is maintained in an expensive controlled facility. As an example, assuming a liver weight to 1.5 % of total bodyweight, a foetal yak weighing 5 kg would yield 75 grams of liver derived matrix source material. Hence production costs of yak-based matrix are much lower as compared to conventional Matrigel®. A less expensive organoid growth sustaining substrate would be an advantage to the biomedical research community fostering developing better treatment of disease amongst other beneficial effects.

[0029] In addition, the availability of such a product would be ethically desirable. Whereas the mice used for Matrigel® production suffer from the outrageous discomfort from carrying tumours that constitute 25% of bodyweight while living in confined conditions, the livers from foetal yaks can be obtained from semi-wild vast rangeland grazing areas of the Qinghai-Tibetan Plateau from the non-viable calves resulting from the spontaneous matings between Bos Grunniens and Bos Taurus, calves which otherwise would have little value for subsistence farmers herding the animals. Hence, switching by the biomedical research community from conventional Matrigel® to the ECM extract of the present invention would be accompanied by a substantial reduction in animal suffering, while life of very poor populations that live from the free-ranging yak herds would improve as well.

[0030] A further consideration might be environmental impact. Mice for Matrigel® production are typically housed in laboratory animal facilities that require at least twenty ach (air changes per hour), ventilation requirements that are associated with intensive energy and public water source water use and thus CO2 footprint. Conversely, the yak calves serving as the source material do not contribute to global CO2 emissions apart from that associated with the transport of the dead calf to the processing facility.

[0031] Finally, ECM extracts and hydrogels of the present invention may also support medical organoid applications, wherein e.g. 3D-organoid culture are used as alternatives for organ transplantation and regenerative medicine. Such in vivoapplications are not possible with organoids cultured using Matrigel® because of the sarcoma-derived origin of this substrate.

[0032] In a first aspect, the invention therefore provides a method for preparing an extracellular matrix (ECM) extract, comprising:

[0033] - providing organ tissue of an animal of the genus Bos,

[0034] - decellularizing the organ tissue, and

[0035] - digestion of the decellularized organ tissue.

[0036] Further provided is an ECM extract or hydrogel obtainable or obtained by a method according to the invention. Further provided is an extracellular matrix (ECM) extract, hydrogel or derivative thereof derived from organ tissue of an animal of the genus Bos, preferably a foetal or neonatal animal, more preferably a foetal or still-born foetal animal. Also provided is a composition comprising an ECM extract or hydrogel according to the invention.

[0037] The ECM is an intricate network composed of extracellular macromolecules and minerals, that surround and support cells and tissues in the human an animal body, and which is organized in a cell / tissue-specific manner. Major components include collagens, proteoglycans, elastin, and cell-binding glycoproteins.

[0038] An ECM extract or hydrogen of the invention is prepared from organ tissue of an animal of the species Bos. Said organ can be any organ that comprises ECM components, in particular ECM proteins. ECM in the organ of animals may include interstitial matrices and basement membranes. Consequently, an ECM extract or hydrogel of the invention may comprise components thereof. Non-limiting examples of organ tissue include liver tissue, skin tissue, kidney tissue, lung tissue, heart tissue, muscle tissue, stomach tissue, pancreatic tissue, lymph nodes, thymus, tendon tissue, bone tissue, intestine tissue, brain tissue, bladder tissue, genital organ tissue, breast tissue, extra-embryonic tissue (including placenta and yolk sac) and combinations thereof. In preferred embodiments, the organ tissue is or comprises liver tissue, kidney tissue, skin tissue, and / or intestine tissue. In some preferred embodiments, said organ tissue is or comprises liver tissue. In some embodiments, the organ tissue is an organ, which allows perfusion of the organ in the method of preparing an ECM extract or hydrogel of the invention. For instance, liver or liver tissue can be perfused through the hepatic artery and / or the portal vein. As another example, kidney or kidney tissue can be perfused through the renal artery.

[0039] An ECM extract of the present invention or prepared in accordance with the present invention is a complex mixture of ECM proteins. Such ECM extract resultsfrom the decellularization and digestion of organ tissue. It can be in any form, e.g. the decellularized and digested tissue as such, in powder form after dehydration by e.g. lyophilization, or in the form of a reconstituted powder or pre-gel solution. Thus, an ECM extract or composition of the invention can be in the form of a powder, a solution, or a gel.

[0040] In preferred embodiments, an ECM extract of the invention is or is used to prepare a hydrogel. Hence, a method of the invention is preferably for preparing a hydrogel. A hydrogel in the context of the present invention is an ECM -based hydrogel, which means that the hydrogel is the result of the gelation of ECM proteins. In preferred embodiments, a method of the invention is therefore for preparing an ECM based hydrogel and further comprises allowing the ECM extract, in particular the decellularized and digested organ tissue, optionally in the form of a solution of a reconstituted powder, to form a hydrogel. As used herein the term “hydrogel” refers to a hydrated polymeric network. The polymers are crosslinked by covalent, ionic, or hydrogen bonds to result in a three-dimensional porous structure that encapsulates water molecules forming a gel.

[0041] An ECM extract or hydrogel of the invention is composed of a mixture of proteins, in particular ECM derived proteins. Examples of such proteins include, but are not limited to, collagen, laminin, nidogen-1, and proteoglycans. Further examples of proteins and proteoglycans that may be present in an ECM extract or hydrogel of the invention include LAMA1, LAMC1, LAMB1, NIDI, HSPG2, COL4A1, NID2, COL4A2, PXDN, FGG, COL1A1, COL1A2, COL3A1, COL5A2, COL5A1, COL6A3, COL6A2, COL6A1, COL2A1, and / or COL5A3. Yet further examples of proteins and proteoglycans that may be present include periostin, fibrinogen, fibronectin, perlecan, tenascin, EMILIN, lumican elastin, titin, perilipin, dermatopontin, vitronectin, antitrypsin, and fibulin. In preferred embodiments, an ECM extract or ECM hydrogel of the invention comprises one or more protein selected from the group consisting of collagen, laminin, nidogen-1, and proteoglycan. In further preferred embodiments, the ECM extract or ECM hydrogel of the invention comprises collagen and laminin, more preferably collagen, laminin and nidogen-1, more preferably collagen, laminin, nidogen-1, and proteoglycan.

[0042] In some preferred embodiments, an ECM extract or hydrogel of the invention comprises one or more of collagen type I, II, III, IV, V and XI. In some preferred embodiments, collagen type I and III are most abundant. In some preferred embodiments, the collagen comprises COL1A1, COL1A2, COL3A1, COL5A2, COL5A1, COL6A3, COL6A2, COL6A1, COL2A1, and / or COL5A3. In some preferred embodiments, the collagen comprises COL1A2, COL6A2, COL5A1,COL5A2, COL5A3, COL6A1 and combinations thereof. In some preferred embodiments, the collagen comprises COL1A2, COL6A2, COL5A1, COL5A2, COL5A3, and COL6A1.

[0043] An exemplary ECM extract or hydrogel of the invention may comprise 50-95% collagen, in particular including collagen type I and III, preferably comprises 60-95% collagen, more preferably 70-95% collagen more preferably 85-95% collagen more preferably 87-93% collagen. The percentage refers to the content of collagen as weight percentage of the total protein content of the ECM extract or hydrogel. In some preferred embodiments, an ECM extract or hydrogel of the invention comprises 50-95% collagen, in particular including collagen type I and III, more preferably 50-80%, more preferably 60-80%. Further, an ECM extract or hydrogen of the invention may comprise less than 40% laminin, preferably less than 35% laminin, less than 30% laminin, less than 25% laminin, less than 20% laminin, less than 15% laminin, less than 10% laminin, less than 5% laminin. In some preferred embodiments, an ECM extract or hydrogen of the invention comprises less than 15% laminin. In some preferred embodiments, an ECM extract or hydrogen of the invention comprises less than 10% laminin. In some preferred embodiments, an ECM extract or hydrogen of the invention comprises less than 5% laminin. The percentage refers to the content of laminin as weight percentage of the total protein content of the ECM extract or hydrogel.

[0044] ECM proteins, including collagen, laminin, nidogen-1, and proteoglycan, are highly conserved. However, different species show difference at the amino acid level in the sequence of these proteins. An ECM extract or hydrogel of the invention or prepared in accordance with the invention comprises protein derived from an animal of the genus Bos. As used herein the terms “derived from” in this context, including the term “yak-derived” and similar terms, mean that the protein is the specific ortholog of the protein of the indicated animal or species. In preferred embodiments, an ECM extract or hydrogel comprises yak-derived protein, either Bos grunniens or Bos mutus derived protein, preferably Bos grunniens derived protein. Since the organ tissue is preferably derived from a hybrid of a yak and a cow, such as a dairy cow or zebu, the ECM extract or hydrogel may further comprise protein derived from an animal of the species Bos, other than a yak, such as dairy cow (Bos taurus) derived protein or zebu (Bos indicus) derived protein. In some preferred embodiments, an ECM extract or hydrogel comprises both yak-derived protein and cow-derived protein, preferably Bos grunniens or Bos mutus derived protein and Bos taurus or Bos indicus derived protein.Said protein derived from an animal of the species Bos, in particular a yak-derived protein, is preferably selected from the group consisting of collagen, laminin, nidogen-1, proteoglycan and combinations thereof. Preferably said protein comprises collagen, more preferably collagen type I and / or III.

[0045] In particularly preferred embodiments, an ECM extract or hydrogel of the invention comprises yak-derived collagen, yak-derived laminin, yak-derived nidogen-1 and / or yak-derived proteoglycan, in particular comprises yak-derived collagen, in particular yak-derived collagen type I and / or yak-derived collagen type III. Preferably said yak is a Bos grunniens or Bos mutus. In one preferred embodiment, an ECM extract or hydrogel of the invention comprises yak-derived collagen, in particular yak-derived collagen type I and / or yak-derived collagen type III.

[0046] In further preferred embodiments, an ECM extract or hydrogel of the invention comprises cow-derived collagen, cow-derived laminin, and / or cow-derived nidogen-1, preferably wherein the cow is Bos taurus or Bos indicus. In some preferred embodiments, an ECM extract or hydrogel comprises both yak-derived collagen, yak- derived laminin, yak- derived nidogen-1 and / or yak-derived proteoglycan, in particular comprises yak-derived collagen, and comprises cow-derived collagen, cow-derived laminin, and / or cow-derived nidogen-1, in particular comprises cow-derived collagen, preferably wherein the yak is Bos grunniens or Bos mutus and the cow is Bos taurus or Bos indicus.

[0047] In preferred embodiments, the yak-derived collagen comprises an amino acid sequence selected from the sequences of the following proteins:

[0048] AAR82961.1: collagen type I alpha 2, partial [Bos grunniens], XP_070236310.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070213913.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070238891.1: collagen, type I, alpha la-like [Bos mutus], XP_005890387.2: collagen alpha- 1(1) chain [Bos mutus], XP_005909757.1: collagen alpha-2(I) chain [Bos mutus],

[0049] ELR60286.1: Collagen alpha-l(I) chain [Bos mutus],

[0050] XP_005889602.1: collagen alpha- 1 (III) chain [Bos mutus],

[0051] ELR61708.1 Collagen alpha- l(XXIV) chain, partial [Bos mutus], ELR60869.1 Collagen alpha-2(V) chain [Bos mutus],

[0052] ELR60453.1 Collagen type IV alpha-3-binding protein [Bos mutus], ELR60401.1 Collagen alpha-4(IV) chain, partial [Bos mutus], ELR58589.1 Collagen alpha-2(VIII) chain, partial [Bos mutus], ELR58439.1 Collagen alpha- l(XXVI) chain, partial [Bos mutus], ELR57599.1 Collagen alpha-3(IX) chain [Bos mutus],

[0053] ELR57484.1 Collagen alpha- 1(IX) chain, partial [Bos mutus],ELR56035.1: Collagen alpha-l(XXV) chain [Bos mutus], ELR55613.1: Collagen alpha-l(VIII) chain [Bos mutus],

[0054] ELR55477.1: Collagen alpha-l(XV) chain, partial [Bos mutus], ELR55169.1: Collagen alpha-l(V) chain, partial [Bos mutus], ELR54817.1: Collagen alpha-l(XI) chain [Bos mutus],

[0055] ELR54507.1: Collagen alpha-5(VI) chain, partial [Bos mutus], ELR53805.1: Collagen triple helix repeat-containing protein 1, partial [Bos mutus],

[0056] ELR52129.1 Collagen alpha- 1 (XXVII) chain, partial [Bos mutus], ELR51625.1 Collagen alpha- 1 (VI) chain [Bos mutus],

[0057] ELR51623.1 Collagen alpha-2(VI) chain [Bos mutus],

[0058] ELR51256.1 Collagen alpha- 1 (XII) chain, partial [Bos mutus], ELR50313.1 Collagen alpha- 1 (XXII) chain, partial [Bos mutus], ELR49862.1 Collagen alpha- 1 (IV) chain, partial [Bos mutus], ELR49861.1 Collagen alpha-2(IV) chain, partial [Bos mutus], ELR49258.1 Collagen alpha- 1(11) chain, partial [Bos mutus], ELR48724.1 Collagen alpha- l(XVI) chain [Bos mutus],

[0059] ELR46933.1 Collagen alpha-l(XIV) chain, partial [Bos mutus], ELR46375.1 Collagen alpha- 1 (VII) chain [Bos mutus],

[0060] ELR46121.1 Collagen alpha-2(I) chain [Bos mutus],

[0061] ELR45494.1 Collagen alpha- 1 (XXVIII) chain, partial [Bos mutus], ELR45179.1 Collagen alpha-6(IV) chain, partial [Bos mutus],

[0062] ELR45178.1 Collagen alpha-5(IV) chain, partial [Bos mutus], ELR44952.1 Collagen alpha- 1(XX) chain, partial [Bos mutus], ELR44506.1 Collagen alpha-4(VI) chain, partial [Bos mutus], and combinations thereof,

[0063] or a sequence that has at least 90% sequence identity to any of these sequences, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 93%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92% sequence identity to any of these sequences.

[0064] In further preferred embodiments, the yak-derived collagen comprises an amino acid sequence selected from the sequences of the following proteins:

[0065] AAR82961.1: collagen type I alpha 2, partial [Bos grunniens], XP_070236310.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070213913.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070238891.1: collagen, type I, alpha la-like [Bos mutus], XP_005890387.2: collagen alpha- 1(1) chain [Bos mutus], XP_005909757.1: collagen alpha-2(I) chain [Bos mutus],ELR60286.1: Collagen alpha-l(I) chain [Bos mutus],

[0066] XP_005889602.1: collagen alpha- 1 (III) chain [Bos mutus], and combinations thereof,

[0067] or a sequence that has at least 90% sequence identity to any of these sequences, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 93%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92% sequence identity to any of these sequences. The amino acid sequences of AAR82961.1, XP_070236310.1, XP_070213913.1, XP_070238891.1, XP_005890387.2, XP_005909757.1, ELR60286.1 and XP_005889602.1 are shown in figure 2.

[0068] In preferred embodiments, the yak-derived laminin comprises an amino acid sequence selected from the sequences of the following proteins:

[0069] ELR60350.1: Laminin subunit alpha-4 [Bos mutus],

[0070] ELR58274.1: Laminin subunit gamma-2, partial [Bos mutus], ELR58273.1: Laminin subunit gamma-1, partial [Bos mutus], ELR57607.1: Laminin subunit alpha-5 [Bos mutus],

[0071] ELR55063.1: Laminin subunit alpha-2, partial [Bos mutus], ELR54132.1: Laminin subunit alpha-3, partial [Bos mutus], ELR52343.1: Laminin subunit beta-3 [Bos mutus],

[0072] ELR51295.1: Laminin subunit gamma-3, partial [Bos mutus], ELR46390.1: Laminin subunit beta- 2 [Bos mutus],

[0073] ELR45100.1: Laminin subunit beta-1, partial [Bos mutus], XP_070234933.1 laminin subunit gamma-3 [Bos mutus], XP_070232750.1 laminin subunit alpha-2 [Bos mutus], XP_070232505.1 laminin subunit alpha-4 isoform X2 [Bos mutus], XP_070232504.1 laminin subunit alpha-4 isoform XI [Bos mutus], XP_070232503.1 laminin subunit alpha-4 isoform XI [Bos mutus], XP_005890266.2 laminin subunit alpha-4 isoform XI [Bos mutus], XP_005909429.2 laminin subunit beta-2 [Bos mutus],

[0074] XP_070215061.1 laminin subunit beta-2 [Bos mutus],

[0075] XP_070215060.1 laminin subunit beta-2-like, partial [Bos mutus], XP_070237552.1 laminin subunit alpha- 5 [Bos mutus], XP_070240924.1 laminin subunit gamma-2 [Bos mutus],

[0076] XP_070240820.1 laminin subunit beta- 3 [Bos mutus],

[0077] XP_070240819.1 laminin subunit beta- 3 [Bos mutus],

[0078] XP_070240818.1 laminin subunit beta- 3 [Bos mutus],

[0079] XP_070240817.1 laminin subunit beta- 3 [Bos mutus],

[0080] XP_005910983.1 laminin subunit beta-1 [Bos mutus],XP_005893142.1: laminin subunit gamma-2 [Bos mutus], and combinations thereof,

[0081] or a sequence that has at least 90% sequence identity to any of these sequences, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 93%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92% sequence identity to any of these sequences.

[0082] In preferred embodiments, the yak-derived laminin comprises an amino acid sequence selected from the sequences of the following proteins:

[0083] XP_070220870.1: nidogen-1 isoform X2 [Bos mutus],

[0084] XP_005905707.2: nidogen-1 isoform XI [Bos mutus], and

[0085] a combination thereof,

[0086] or a sequence that has at least 90% sequence identity to any of these sequences, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 93%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92% sequence identity to any of these sequences. The amino acid sequences of XP_070220870.1 and XP_005905707.2 are shown in figure 2.

[0087] The ECM extract and hydrogel of the present invention is derived from organ tissue of an animal of the genus Bos. The animal of the genus Bos is for instance an animal of the subspecies Bos taurus, Bos indicus, Bos gaurus, Bos frontalis, Bos javanicus, Bos mutus, Bos grunniens, Bos bison or Bos bonasus. In preferred embodiments, the animal is a yak (Bos grunniens or Bos mutus) or a cow or a hybrid thereof. The cow is preferably a dairy cow (Bos taurus) or zebu (Bos indicus). In a further preferred embodiment, the animal is a hybrid of a yak (Bos grunniens or Bos mutus) and a cow (Bos taurus or Bos indicus), more preferably of a yak (Bos grunniens or Bos mutus) and a dairy cow (Bos taurus).

[0088] Bos grunniens and Bos mutus are the domestic and wild forms, respectively, of the bovid commonly called the yak. Bos mutus is a large, wild bovine native to the Himalayas. It is the ancestor of the domestic yak (Bos grunniens). Bos mutus inhabits remote high-elevation alpine meadows and alpine steppe in rolling to mountainous terrain in the Tibetan Plateau, and Bos grunniens is maintained in China and other parts of Central Asia.

[0089] In preferred embodiments, the animal is a foetus or a neonatal animal, preferably a foetal or neonatal animal of the subspecies Bos grunniens, Bos mutus, Bos taurus, Bos indicus or foetal or neonatal animal of two of said subspecies. As used herein “foetus” includes still-born foetuses. As used herein “neonatal”preferably refers to a newborn animal up to three months of age, preferably up to two months of age, more preferably up to one month of age, i.e. an animal up to three, two or one month after birth. As used herein, “neonatal” animals can include premature animals and stillborn animals. In preferred embodiments, the animal is a foetal hybrid of a yak (Bos grunniens or Bos mutus) and a cow (Bos taurus or Bos indicus), more preferably of a hybrid of a domestic yak (Bos grunniens) and a dairy cow (Bos taurus). Such hybrids result from the spontaneous matings between Bos Grunniens and Bos Taurus, and are not viable resulting in termination of pregnancy before the foetus is full term and stillbirth of the foetus. Hence, in further preferred embodiments, the animal is a still-born foetus, preferably a foetus of a hybrid of a yak (Bos grunniens or Bos mutus) and a cow (Bos taurus or Bos indicus), more preferably of a hybrid of a domestic yak (Bos grunniens) and a dairy cow (Bos taurus).

[0090] Organ tissue used in accordance with the present invention is thus preferably isolated from the animal of the genus Bos as defined herein above. The organ tissue may be isolated from a single animal or from more than one animal. In some embodiments, the organ tissue is an organ from a single animal. This for instance, may allow the organ to be perfused by solution comprising the component, such as solubilizer, DNase and proteases, used to prepare an ECM extract or hydrogel in accordance with the invention. In some preferred embodiments the organ or organ tissue is liver or liver tissue.

[0091] In some embodiments, yak ECM may be enriched in several proteins as compared to ECM of other animals, including other animals of the species Bos. Examples of such proteins are growth factors like insulin-like growth factor I (IGF-1), erythropoietin (EPO), and bovine somatotropin. Hence, in some embodiments, an ECM extract or hydrogel of the invention comprises IGF-1, EPO and / or bovine somatotropin. In other embodiments, an ECM extract of hydrogel of the invention is enriched in IGF-1, EPO and / or bovine somatotropin, as compared to an ECM extract or ECM hydrogel obtained from the same organ tissue of another animal than yak, in particular another animal of the species Bos, that is prepared with the same method. As used herein “enriched” preferably indicates that the concentration of the relevant protein, e.g. IGF-1, EPO or bovine somatotropin, in an ECM extract or hydrogel of the invention is at least 10% higher than the concentration of the same protein in an ECM extract or ECM hydrogel obtained from the same organ tissue of another animal than yak, in particular another animal of the species Bos, that is prepared with the same method, e.g. at least 20% higher, at least 30% higher, at least 40% higher, or at least 50% higher.In some embodiments, an ECM extract or hydrogel of the invention is essentially devoid of growth factors. The amount of growth factors may be difficult to control and an extract or hydrogel that is devoid of growth factors may aid in increasing reproducibility and reducing batch-to-batch variations.

[0092] A method of the invention comprises decellularizing the organ tissue.

[0093] “Decellularization” as used herein refers to the removal or degradation of cells from the organ tissue. In preferred embodiments, decellularization results in the removal or degradation of at least 90%, more preferably at least 95%, more preferably at least 99% of cells from the organ tissue, as compared to the cells present in the organ tissue before decellularization. Methods for decellularizing organ tissue are well known in the art and a skilled person is well capable of selecting an appropriate method for decellularizing. For instance, decellularization of the organ tissue may comprise treatment of the organ tissue with a surfactant, by sonication, by freeze / thaw cycles and combinations thereof. Nondimiting examples of suitable surfactants are sodium dodecyl sulfate and Triton X-100.

[0094] Decellularization preferably comprises removal or degradation of DNA. In preferred embodiments, decellularization results in the removal of at least 90%, more preferably at least 95%, more preferably at least 99% of the DNA, as compared to the DNA present in the organ tissue sample before decellularization. Methods for DNA removal or degradation are well known in the art and a skilled person is well capable of selecting an appropriate method for DNA removal or degradation. For instance, the decellularization comprises treatment organ tissue with a deoxyribonuclease (DNase). DNases are glycoprotein endonucleases that catalyse the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thereby degrading the DNA. Multiple DNases are known and can be used in a method of the invention. Examples include DNase I, DNaselEl, DNase 1E2, DNase 1E3, DNase II a and DNase II B. In preferred embodiments the DNase is DNase type I. DNase treatment may be performed at the same time as treatment with e.g. a surfactant or subsequent to treatment e.g. a surfactant.

[0095] Decellularization by treatment with a surfactant and optionally treatment with DNase may be performed by incubating the organ tissue, which can optionally e.g. be fractionated, grinded or pulverized, with a solution comprising the surfactant and optionally DNase, or with a solution comprising the surfactant, followed by incubation with a solution comprising DNase. Alternatively, the organ tissue may be perfused with a solution comprising the surfactant and optionally DNase, or perfused with a solution comprising the surfactant, followed by perfusion with a solution comprising DNase. The solution is preferably a buffer, more preferably an aqueous buffer. In preferred embodiments, decellularizationcomprises perfusing the organ tissue with a buffer comprising a surfactant, and optionally treatment with DNase. In further preferred embodiments, decellularization comprises perfusing the organ tissue with a buffer comprising a surfactant, and treatment with DNase.

[0096] A method of the invention comprises digestion of the decellularized organ tissue. “Digestion” as used herein refers to the process by which decellularized tissue is broken down into a smaller fragments and / or components are extracted from the decellularized tissue. Several digestions methods can be used to digest and / or solubilize the decellularized organ tissue. Preferred examples are enzymatic digestion and chemical digestion.

[0097] “Enzymatic digestion” as used herein refers to treatment with a protease to cleave peptide bonds in the protein backbone. Hence, the enzyme is preferably a protease. Protein fragmentation is preferred in order to solubilize the ECM, in particular in powder form, due to the insoluble nature of most ECM proteins, specifically collagen I. Methods for enzymatic digestion of organ tissue are well known in the art and a skilled person is well capable of selecting an appropriate method for enzymatic digestion. Preferred examples of enzymes used in enzymatic digestion include, but are not limited to, trypsin, chymotrypsin, Lys-C, GluC, Arg-C, Asp-N and pepsin. In some preferred embodiments, enzymatic digestion comprises pepsin digestion. Pepsin is a non-specific protease which cleaves peptide bonds in the protein backbone following aromatic residues (i.e., Tryptophan, Tyrosine, Phenylalanine).

[0098] “Chemical digestion” as used herein is also referred to as “chemical extraction” and refers to treatment with one or more compounds that break down components of the decellularized tissue into smaller fragments and / or to extract components from decellularized tissue, e.g. to extract proteins. For instance, collagen can be extracted from decellularized tissue by acidic treatment, using e.g. organic acids (e.g. acetic acid, chloracetic acid, citric acid, lactic acid) or inorganic acid (e.g. hydrochloric acid). As another example, laminin can be extracted from decellularized tissue by treatment with urea. Hence, in preferred embodiments, chemical digestion comprises treatment with an acid, such as acetic acid, chloracetic acid, citric acid, lactic acid and / or hydrochloric acid, and / or treatment with urea.

[0099] In preferred embodiments, the digestion comprises or is enzymatic digestion and / or chemical digestion. In some preferred embodiments, the digestion comprises or is enzymatic digestion. In some preferred embodiments, the digestion comprises or is enzymatic digestion and chemical digestion.Digestion, preferably enzymatic and / or chemical digestion, may be performed following decellularization of the organ tissue. However, in some preferred embodiments, the decellularized organ tissue is, following decellularization, dehydrated to provide a powder, prior to digestion. More preferably, the decellularized organ tissue is processed to smaller fragments, e.g. by cutting into such smaller fragments, optionally fractionated, grinded or pulverized, and dehydrated to provide a powder. Preferably, digestion is subsequently performed by digesting the powder with the protease and / or by chemical treatment.

[0100] Similar to the decellularization step, digestion may be performed by incubating the decellularized organ tissue or powder with a solution comprising the protease, preferably pepsin, and / or compound such as an acid as described herein or urea. Alternatively, the organ tissue, prior to dehydration, may be perfused with a solution comprising the protease, preferably pepsin, and / or compound such as an acid as described herein or urea. The solution comprising the protease, preferably pepsin, and / or compound such as an acid as described herein or urea is preferably a buffer, more preferably an aqueous buffer.

[0101] As indicated herein above, the decellularized organ tissue is preferably dehydrated prior to digestion. More preferably, the decellularized organ tissue is processed to smaller fragments, e.g. by cutting into such smaller fragments, optionally grinded or pulverized, and dehydrated to provide a powder.

[0102] “Dehydration” as used herein refers to a process to remove water, i.e. drying of the decellularized, optionally cut and grinded / pulverized, organ tissue. Examples of dehydration methos include but are not limited to vacuum drying, lyophilizing, and heat or light drying In preferred embodiments, dehydration comprises lyophilization. Lyophilization, also referred to as freeze-drying, is well known in the art. Lyophilization may optionally be followed by fractionation, grinding or pulverization, such as mechanical grinding or pulverization.

[0103] Hence, in preferred embodiments, a method of the invention comprises:

[0104] - providing organ tissue of an animal of the genus Bos,

[0105] - decellularizing the organ tissue,

[0106] - dehydrating the decellularized organ tissue to obtain a powder,

[0107] - digestion, preferably enzymatic and / or chemical digestion, of the powder.

[0108] The steps are performed in the indicated order.

[0109] Following digestion, a mixture is obtained comprising the digested powder. Decellularization and digestion results in an ECM extract according to the invention. In preferred embodiments, the mixture may be reconstituted or diluted in an aqueous buffer to obtain a pre-gel solution. The term “pre-gel solution” refersto a solution comprising an ECM extract of the invention that has not yet formed a gel, in particular hydrogel. The pre-gel solution can be cooled, e.g. to a temperature of 4° C or less, and optionally diluted and aliquoted. The obtained ECM extract, may be stored, preferably at a temperature of 4°C or less, preferably frozen such as at -20 °C, so that a hydrogel can be formed later, or a hydrogel can be formed from the pre -gel solution.

[0110] Hence, in some embodiments, a method of the invention comprises, in the indicated order:

[0111] - providing organ tissue of an animal of the genus Bos,

[0112] - decellularizing the organ tissue,

[0113] - dehydrating the decellularized organ tissue to obtain a powder,

[0114] - digestion, preferably enzymatic and / or chemical digestion, of the powder, - optionally reconstituting or diluting the mixture obtained in the previous step in an aqueous buffer to obtain a pre-gel solution,

[0115] - cooling to a temperature of 4° C or less, and

[0116] - optionally diluting the cooled material to obtain a pre-gel solution.

[0117] A method of the invention, preferably a method for preparing an ECM hydrogel, may further comprise allowing the decellularized and digested organ tissue, reconstituted powder or pre-gel solution, in particular the pre-gel solution to form a hydrogel. The pre-gel solution will form a hydrogel at any temperature above 4°C. However, at such low temperatures, gelling will occur at a very slow rate. The higher the temperature, the shorter the gelling time. In preferred embodiments, the pre-gel solution is allowed to form a hydrogel at a temperature of 12°C or higher, preferably between 12°C and 42°C. Gelling time is advantageously fast at temperatures from room temperature to about 42°C. Hence, in further preferred embodiments, the pre-gel solution is allowed to form a hydrogel at a temperature of 18°C - 42°C or 18°C - 37°C. In some embodiments, the formation of a hydrogel is performed in a humidified CO2 incubator. In preferred embodiments, the hydrogel is maintained in the formed shape for at least 5 days, preferably at least one week. The hydrogel may be maintained for up to 2 weeks or longer.

[0118] The hydrogel can be allowed to form in different shape, such as a spherical or sheet-like shape, which optimally support the cells or tissue that is subsequently cultured on the hydrogel.

[0119] In the experimental section a suitable, exemplary method for preparing an ECM extract and ECM hydrogel is detailed. Figure 1 shows an exemplary workflow of such method. A detailed exemplary method is the following:

[0120] Collecting organ or organ tissue from the animal,Rinsing the organ or organ tissue by perfusion (120 mg Hg) with dHaO, in case of liver via the hepatic artery

[0121] Flushing the organ or organ tissue with an aqueous solution comprising 4% Triton X-100 and 1% NH2 to achieve decellularization, preferably 5 cycles of 120 minutes

[0122] Continuous perfusion with 50 Liters of dLLO

[0123] Storing at 4 degrees Celsius for 10 days in dLLO, which is replaced every 24 hours

[0124] Perfusing the organ or organ tissue with an aqueous solution comprising 5 mg / L DNase type I, 0.9% NaCl, 100 mM CaCL, 100 mM MgCk for 120 min In case of liver or liver tissue: removal of the collagenous Glisson’s capsule Optionally cutting into smaller fragments

[0125] Lyophilizing by freeze-drying of the tissue for 72 hour

[0126] Mechanical pulverization

[0127] Enzymatic digestion, with 40 mg / ml pepsin (3200-4500 U / mg) in 0.5M acetic acid for 72 hours

[0128] Cooled to close to 0 degrees Celsius and resuspended in buffer (PBS and Hepes-buffered advanced DMEM / F12, pH adjusted to 7.5 using NaOH) Dilution to 8 mg / ml to form the pre- gel stock solution

[0129] Stock solution is subjected to centrifugation at 4 degrees C to remove undigested debris

[0130] Plating the material in tissue culture plates in a humidified CO2 incubator at 37 degrees C for 30 minutes and upside down rotation results in gelation.

[0131] The invention thus provides an extracellular matrix (ECM) extract or derivative thereof, in particular derived from organ tissue of an animal of the genus Bos, in particular a foetal, still-born foetal or neonatal animal, comprising collagen, laminin, and / or nidogen-1 derived from an animal of the genus Bos. The invention further provides an ECM-based hydrogel or derivative thereof, in particular derived from organ tissue of an animal of the genus Bos, in particular a foetal, still-born foetal or neonatal animal, comprising collagen, laminin, and / or nidogen-1 derived from an animal of the genus Bos.

[0132] The animal of the genus Bos is for instance an animal of the subspecies Bos taurus, Bos indicus, Bos gaurus, Bos frontalis, Bos javanicus, Bos mutus, Bos grunniens, Bos bison or Bos bonasus. In preferred embodiments, the animal is a yak (Bos grunniens or Bos mutus) or a cow or a hybrid thereof. The cow is preferably a dairy cow (Bos taurus) or zebu (Bos indicus). In a further preferred embodiment, the animal is a hybrid of a yak (Bos grunniens or Bos mutus) and acow (Bos taurus or Bos indicus), more preferably of a yak (Bos grunniens or Bos mutus) and a dairy cow (Bos taurus).

[0133] In preferred embodiments, the ECM extract, hydrogel or derivative thereof comprises yak-derived collagen, yak-derived laminin, and / or yak-derived nidogen-1, preferably at least yak-derived collagen. In preferred embodiments, the ECM-based hydrogel comprises yak-derived collagen, yak-derived laminin, and / or yak-derived nidogen-1.

[0134] In further preferred embodiments, the ECM extract, hydrogel or derivative thereof of the invention comprises yak-derived collagen, yak-derived laminin, yak-derived nidogen-1 and / or yak-derived proteoglycan, in particular comprises yak-derived collagen, in particular yak-derived collagen type I and / or yak-derived collagen type III. Preferably said yak is a Bos grunniens or Bot mutus.

[0135] In preferred embodiments, the yak-derived collagen comprises an amino acid sequence selected from the sequences of the following proteins:

[0136] AAR82961.1: collagen type I alpha 2, partial [Bos grunniens], XP_070236310.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070213913.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070238891.1: collagen, type I, alpha la-like [Bos mutus], XP_005890387.2: collagen alpha- 1(1) chain [Bos mutus], XP_005909757.1: collagen alpha-2(I) chain [Bos mutus],

[0137] ELR60286.1: Collagen alpha-l(I) chain [Bos mutus],

[0138] XP_005889602.1: collagen alpha- 1 (III) chain [Bos mutus],

[0139] ELR61708.1 Collagen alpha- l(XXIV) chain, partial [Bos mutus], ELR60869.1 Collagen alpha-2(V) chain [Bos mutus],

[0140] ELR60453.1 Collagen type IV alpha-3-binding protein [Bos mutus], ELR60401.1 Collagen alpha-4(IV) chain, partial [Bos mutus], ELR58589.1 Collagen alpha-2(VIII) chain, partial [Bos mutus], ELR58439.1 Collagen alpha- l(XXVI) chain, partial [Bos mutus], ELR57599.1 Collagen alpha-3(IX) chain [Bos mutus],

[0141] ELR57484.1 Collagen alpha- 1(IX) chain, partial [Bos mutus], ELR56035.1 Collagen alpha- 1 (XXV) chain [Bos mutus],

[0142] ELR55613.1 Collagen alpha- 1 (VIII) chain [Bos mutus],

[0143] ELR55477.1 Collagen alpha- 1(XV) chain, partial [Bos mutus], ELR55169.1 Collagen alpha- 1(V) chain, partial [Bos mutus], ELR54817.1 Collagen alpha- 1 (XI) chain [Bos mutus],

[0144] ELR54507.1 Collagen alpha-5(VI) chain, partial [Bos mutus], ELR53805.1 Collagen triple helix repeat-containing protein 1, partial [Bos mutus],

[0145] ELR52129.1: Collagen alpha- 1 (XXVII) chain, partial [Bos mutus],ELR51625.1 Collagen alpha- 1 (VI) chain [Bos mutus],

[0146] ELR51623.1 Collagen alpha-2(VI) chain [Bos mutus],

[0147] ELR51256.1 Collagen alpha- 1 (XII) chain, partial [Bos mutus], ELR50313.1 Collagen alpha- 1 (XXII) chain, partial [Bos mutus], ELR49862.1 Collagen alpha- 1 (IV) chain, partial [Bos mutus], ELR49861.1 Collagen alpha-2(IV) chain, partial [Bos mutus], ELR49258.1 Collagen alpha- 1(11) chain, partial [Bos mutus], ELR48724.1 Collagen alpha- l(XVI) chain [Bos mutus],

[0148] ELR46933.1 Collagen alpha-l(XIV) chain, partial [Bos mutus], ELR46375.1 Collagen alpha- 1 (VII) chain [Bos mutus],

[0149] ELR46121.1 Collagen alpha-2(I) chain [Bos mutus],

[0150] ELR45494.1 Collagen alpha- 1 (XXVIII) chain, partial [Bos mutus], ELR45179.1 Collagen alpha-6(IV) chain, partial [Bos mutus],

[0151] ELR45178.1 Collagen alpha-5(IV) chain, partial [Bos mutus], ELR44952.1 Collagen alpha- 1(XX) chain, partial [Bos mutus], ELR44506.1 Collagen alpha-4(VI) chain, partial [Bos mutus], and combinations thereof,

[0152] or a sequence that has at least 90% sequence identity to any of these sequences, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 93%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92% sequence identity to any of these sequences.

[0153] In further preferred embodiments, the yak-derived collagen comprises an amino acid sequence selected from the sequences of the following proteins:

[0154] AAR82961.1: collagen type I alpha 2, partial [Bos grunniens], XP_070236310.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070213913.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070238891.1: collagen, type I, alpha la-like [Bos mutus], XP_005890387.2: collagen alpha- 1(1) chain [Bos mutus], XP_005909757.1: collagen alpha-2(I) chain [Bos mutus],

[0155] ELR60286.1: Collagen alpha-l(I) chain [Bos mutus],

[0156] XP_005889602.1: collagen alpha- 1 (III) chain [Bos mutus], and combinations thereof,

[0157] or a sequence that has at least 90% sequence identity to any of these sequences, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 93%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92% sequence identity to any of these sequences. The amino acid sequences of AAR82961.1, XP_070236310.1, XP_070213913.1, XP_070238891.1,XP_005890387.2, XP_005909757.1, ELR60286.1 and XP_005889602.1 are shown in figure 2.

[0158] In preferred embodiments, the yak-derived laminin comprises an amino acid sequence selected from the sequences of the following proteins:

[0159] ELR60350.1: Laminin subunit alpha-4 [Bos mutus],

[0160] ELR58274.1: Laminin subunit gamma-2, partial [Bos mutus], ELR58273.1: Laminin subunit gamma-1, partial [Bos mutus], ELR57607.1: Laminin subunit alpha-5 [Bos mutus],

[0161] ELR55063.1: Laminin subunit alpha-2, partial [Bos mutus], ELR54132.1: Laminin subunit alpha-3, partial [Bos mutus], ELR52343.1: Laminin subunit beta-3 [Bos mutus],

[0162] ELR51295.1: Laminin subunit gamma-3, partial [Bos mutus], ELR46390.1: Laminin subunit beta- 2 [Bos mutus],

[0163] ELR45100.1: Laminin subunit beta-1, partial [Bos mutus], XP_070234933.1 laminin subunit gamma-3 [Bos mutus], XP_070232750.1 laminin subunit alpha-2 [Bos mutus], XP_070232505.1 laminin subunit alpha-4 isoform X2 [Bos mutus], XP_070232504.1 laminin subunit alpha-4 isoform XI [Bos mutus], XP_070232503.1 laminin subunit alpha-4 isoform XI [Bos mutus], XP_005890266.2 laminin subunit alpha-4 isoform XI [Bos mutus], XP_005909429.2 laminin subunit beta-2 [Bos mutus], XP_070215061.1 laminin subunit beta-2 [Bos mutus],

[0164] XP_070215060.1 laminin subunit beta-2-like, partial [Bos mutus], XP_070237552.1 laminin subunit alpha- 5 [Bos mutus], XP_070240924.1 laminin subunit gamma-2 [Bos mutus],

[0165] XP_070240820.1 laminin subunit beta- 3 [Bos mutus], XP_070240819.1 laminin subunit beta- 3 [Bos mutus], XP_070240818.1 laminin subunit beta- 3 [Bos mutus],

[0166] XP_070240817.1 laminin subunit beta- 3 [Bos mutus], XP_005910983.1 laminin subunit beta-1 [Bos mutus], XP_005893142.1 laminin subunit gamma-2 [Bos mutus], and combinations thereof,

[0167] or a sequence that has at least 90% sequence identity to any of these sequences, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 93%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92% sequence identity to any of these sequences.

[0168] In preferred embodiments, the yak-derived laminin comprises an amino acid sequence selected from the sequences of the following proteins:XP_070220870.1: nidogen-1 isoform X2 [Bos mutus],

[0169] XP_005905707.2: nidogen-1 isoform XI [Bos mutus], and

[0170] a combination thereof,

[0171] or a sequence that has at least 90% sequence identity to any of these sequences, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 93%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92%, more preferably at least 92% sequence identity to any of these sequences. The amino acid sequences of XP_070220870.1 and XP_005905707.2 are shown in figure 2.

[0172] In preferred embodiments, an ECM extract or ECM-based hydrogel or derivative thereof of the invention further comprises IGF-1, EPO and / or bovine somatotropin. In further preferred embodiments, an ECM extract, hydrogel or derivative thereof of the invention is enriched in IGF-1, EPO and / or bovine somatotropin, as compared to an ECM extract, hydrogel or derivative thereof obtained from the same organ tissue of another animal than yak, in particular another animal of the species Bos, that is prepared with the same method.

[0173] Preferably, the concentration of the relevant protein, e.g. IGF-1, EPO or bovine somatotropin, in an ECM extract, hydrogel or derivative thereof of the invention is at least 10% higher than the concentration of the same protein in an ECM extract, hydrogel or derivative thereof obtained from the same organ tissue of another animal than yak, in particular another animal of the species Bos, that is prepared with the same method, more preferably at least 20% higher, more preferably at least 30% higher, more preferably at least 40% higher, more preferably at least 50% higher.

[0174] An ECM extract, hydrogel or derivative thereof according to the invention is particularly suitable for use as a substrate for culturing cells, tissue or organoids. Organoids are miniature, simplified versions of organs grown in the lab from stem cells. They mimic key structural and functional features of real human organs, such as the brain, intestine, liver, or kidney — but on a much smaller scale.

[0175] Provided is therefore a use of an ECM extract, hydrogel or derivative thereof according to the invention as a substrate for cell, tissue or organoid culturing. Also provided is a cell, tissue or organoid culture substrate comprising an ECM extract or hydrogel according to the invention. Further provided is a method for culturing cells comprising providing an ECM extract, hydrogel or derivative thereof according to the invention and culturing cells. In preferred embodiments, the cultured cells form a multicellular tissue or organoid.The specific cell, tissue or organoid may be, but is not necessarily, cultured on a substrate of the invention that is prepared from the same organ as the organ from which the cell, tissue or organoid is derived or is intended to mimic. E.g. hepatic cell, tissue or organoids may be cultured on a substrate of the invention that is prepared from liver tissue, but may also be cultured on a substrate that is prepared from other organ tissue.

[0176] The ECM extract, hydrogel or derivative thereof can be used as a substrate for two-dimensional (2D) or three-dimensional (3D) culturing.

[0177] In some preferred embodiments, the cultured cells or tissues are primary cells, preferably the cells are primary tissue cells. Primary cells are cells that are isolated directly from tissues or blood. For example, these primary cells can be hepatocytes, epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic, and / or mesenchymal stem cells. The cultures can be heterogeneous. I.e. the cell culture can also be used to co-culture different cell types. In some embodiments, the cells cultured on the three-dimensional cell, tissue or organoid culture substrate are hepatocytes, epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic and / or mesenchymal stem cells. In some embodiments, the cultures are heterogeneous, comprising various cell types.

[0178] Furthermore, primary cells can be derived from healthy or diseased tissue, for example, from cancerous tissue or tumors. These cells can be cancer or tumor cells but also cells that are present in the microenvironment of a tumor and support the tumor cells.

[0179] The ECM extract, hydrogel or derivative thereof and cell, tissue or organoid culture substrate according to the invention can support the maintenance, growth, survival and differentiation of cells, tissues and organoids, in particular the maintenance, growth and differentiation of organoids and the adherence and the maintenance, growth and differentiation of cell and tissue cultures. In a preferred embodiment of the method for culturing cells and uses of the invention, tissues or organoids as described herein, the cells are stem cells, preferably mesenchymal stem cells, adult stem cells, adipose adult stem cells and / or induced pluripotent stem cells. In some embodiments, the cells are progenitor cells. In preferred embodiments, the stem cells are not derived from embryos or embryonic tissue. Preferably, the stem cells are not embryonic stem cells.

[0180] The ECM extract, hydrogel or derivative thereof and cell, tissue or organoid culture substrate according to the invention are further advantageously used forculturing and maintain cells, tissues and organoids in an organ-on-chip device, a bioprinting device for three-dimensional culture of mammalian cells and in a bioreactor for expansion, the maintenance, growth, and differentiation in organoids at a scale of at least 106cells and / or a hydrogel volume of at least 100 mL. In some embodiments, provided is therefore a use or method of the invention wherein the ECM extract, hydrogel or derivative thereof of the invention is used in an organ-on-chip device or bioprinting device. Three-dimensional bioprinting refers to the use of 3D printing-like techniques to combine cells, growth factors, bio-inks, and biomaterials to form 3D structures, including organoids, and finds use in tissue engineering, regenerative medicine and research into diseases. A collection of cells, such as an organoid, can be printed in combination with an ECM extract or hydrogel of the invention, wherein the ECM extract or hydrogel serves as a scaffold for the printed organoids and supports organoid survival, growth and differentiation. In some embodiments, provided is therefore a use or method of the invention wherein the ECM extract, hydrogel or derivative thereof of the invention is used in a bioreactor for expansion, the maintenance, growth, and differentiation in organoids at a scale of at least 106cells and / or a hydrogel volume of at least 100 mL.

[0181] In preferred embodiments, the cell culture template, as described herein, can be used to grow or create functional 3D structures. In some embodiments, cells in the method for culturing as described herein form complex cellular assemblies, preferably a multicellular organoid. The term “organoid” is well known in the art and refers to a three-dimensional cell tissue that mimics or resembles the functional and / or histological structure of an organ or part thereof. An organoid is essentially a miniaturized and simplified version of an organ produced in vitro in three dimensions. These organoids are multicellular and show realistic microanatomy. They are derived from one or a few cells from a tissue, stem cell, or introduced pluripotent stem cell. Examples of organoids that can be cultured using an ECM extract, hydrogel or derivative thereof according to the invention are intestinal organoids, hepatic organoids, pancreatic organoids, gastric organoids, oesophageal organoids, lung organoids, skin organoids, kidney organoids, and neural organoids.

[0182] Cells are preferably seeded onto or into the ECM extract, hydrogel or derivative thereof of the invention. In vitro culturing of cells, tissues and organoids requires the supply of medium and nutrients. The culture environment should be stable in terms of pH, oxygen supply, and temperature. Cell culture media often comprise balanced salt solutions, amino acids, vitamins, fatty acids and lipids tosupport the growth of the cells, tissues and / or organoids. The precise media formulations have often been derived by optimizing the concentrations of every constituent. Different cell types are in need of different media compositions.

[0183] Furthermore, culturing of cells often requires the addition of serum. The serum is a complex mix of proteins, peptides, growth factors, and growth inhibitors. The most commonly used serum is fetal calf serum, which is used for a wide range of cell types. In addition, the medium may be supplemented with growth factors and cytokines. During culturing, the cells use the nutrients supplied by the media and excrete their waste products into the media. Therefore, it is important to supply the cultured cells or tissues with fresh media regularly. The frequency of refreshing the media depends on the cell type and growth rate of the cells.

[0184] Cells, tissues and organoids cultures using a substrate according to the invention may have various applications. For instance, culturing stem cells on the substrate and inducing tissue-specific cell expansion and organoid formation is useful for regenerative medicine. Regenerative medicine seeks to replace tissue or (partial) organs that have been damaged by e.g. disease, trauma, or congenital origin, replacing or restoring damaged cell, tissue, or organ function. Other applications include the use of the cells, tissues or organoids as a model for studying, e.g. properties of cells, tissues or organs, including as a disease model, such as for cancer studies, and for screening potential therapeutic molecule, including anti-cancer drug screening, and in preclinical models, such in as liver toxicity organoid preclinical models and drug-induced liver injury (DILI) preclinical models.

[0185] Features maybe described herein as part of the same or separate aspects or embodiments of the present invention for the purpose of clarity and a concise description. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

[0186] The invention will be explained in more detail in the following, non-limiting examples.

[0187] Brief description of the drawings

[0188] Figure 1: Exemplary workflow of the production of a hydrogel of the invention.Figure 2: Amino acid sequences of AAR82961.1: collagen type I alpha 2, partial [Bos grunniens], XP_070236310.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070213913.1: collagen, type I, alpha Ib-like [Bos mutus], XP_070238891.1: collagen, type I, alpha la-like [Bos mutus], XP_005890387.2: collagen alpha- 1(1) chain [Bos mutus], XP_005909757.1: collagen alpha-2(I) chain [Bos mutus], ELR60286.1: Collagen alpha-l(I) chain [Bos mutus] and XP_005889602.1: collagen alpha- 1 (III) chain [Bos mutus]

[0189] Figure 3: Amino acid sequences of XP_070220870.1: nidogen-1 isoform X2 [Bos mutus] and XP_070220870.1: nidogen-1 isoform XI [Bos mutus].

[0190] Figure 4: Quantitative analysis of the content of three separate batches of Matriarch, a batch of BME and two batches of Matrigel, integrated for important ECM constituents.

[0191] Figure 5: Routine growth from a intrahepatic cholangiocyte-derived organoid in BME, the current standard and in Matriarch (labelled Yak in the figure).

[0192] Figure 6: A. Experimental set up of the experiment for organoid initiation from liver biopsies. B. Depiction of the results of the upper and lower arm of the experiment.

[0193] Figure 7: Matriarch and Matrigel have distinct properties with respect to organoid phenotype. Shown are distinctive examples of the morphology observed with Matriarch, an example of the reference (Matrigel) and a quantification of the morphological effects seen.

[0194] Figure 8: Distinct differences in gene expression are detected in ICO grown in Matriarch and Matrigel. Shown are distinctive examples of gene expression of a selected panel of genes tested on ICO (N=6).

[0195] Examples

[0196] Example 1 Preparation of a vak / cow derived hydrogel

[0197] Figure 1 shows an exemplary workflow of the production of a hydrogel of the invention. Upon collection of a dead hybrid yak-cow calf and removal of its blood, the liver (approximately 300 grams) is dissected from the foetus, can be frozen and thawed, and is rinsed by continuous pressure-controlled (120 mm Hg (I)) perfusion with 20 L of dl CO applied via the hepatic artery. The same mode is used for the subsequent decellularization with five cycles of flushing the liver with 10 L of a solution containing 4% Triton X-100 and 1 % of NH2, each cycle lasting 120 minutes. Subsequently, the liver remnants are continuously perfused with 50 L of d I CO before being stored at 4°C for 10 days in 10 L of dEFO, replaced by a fresh storage solution every 24 hours. Decellularization is completed by perfusing theliver remnants with a DNase solution of 5 mg / L DNase type I in 0.9% NaCl + 100 mM CaCh + 100 mM MgCk for 120 min. For subsequent production of the powder phase of the new product the bovine collagenous Glisson's capsule is mechanically removed and the remaining foetal liver matrix is cut in smaller fragments and lyophilised trough freeze-drying for 72 hours and mechanically pulverised. The resulting foetal yak liver matrix powder is then digested using 40 mg / ml Pepsin (3.200-4.500 U / mg) in 0.5 M Acetic Acid for 72-hours at ambient temperature. Subsequently, the mixtures is cooled to close to 0 °C and using concentrated stock solutions taken up in PBS and Hepes-buffered advanced DMEM / F12, pH being adjusted to 7.5 employing NaOH. The digested foetal yak matrix is diluted to 8 mg / ml to form the pre-gel stock solution, which is subjected to centrifugation at 4 °C to remove undigested debris. Gelation of the resulting product is evident from plating batches of pre-gel solution in tissue culture plates in a humidified CO2 incubator at 37 °C for 30 min and subsequent upside down rotation of the plates involved. The pre-gel solution, now called matriyak, can be aliquoted and used directly or stored at -20 °C. The resulting matriyak supports proliferation and phenotype maintenance of a variety of organoid models and transferring cultures from Matrigel® and Cultrex® Basement Membrane Extract (BME) to matriyak does not significantly alter the expression of tissue type defining genes, such as the cholangiocyte marker cytokeratin-7 for cholangiocyte organoids, and can support organoid growth and upon appropriate culture condition differentiation of biological material derived from a variety of mammalian species. Moreover, matriyak extracts support the expansion of the iconic intestinal cell organoid culture in a dynamic culture set up without the need for laborious static culture of organoids in hydrogel domes. Overall, the product shows superior characteristics as compared to the existing commercial products available.

[0198] Example 2 Evaluation of composition, batch consistency and organoid initiation

[0199] Matrigel® and BME products are derived from grafted murine sarcoma Englebreth-Holm- Swarm (EHS) mouse cells, whereas a hydrogel prepared in Example 1, also herein referred to as “Matriarch” is derived from foetal / neonatal Bos organ tissue. These fundamentally different biological starting materials and manufacturing routes are expected to yield partially equivalent products but substantially different ECM compositions resulting in alternative functional profiles, which is supported by the proteomics / cell culture evidence summarised below.Material and methods

[0200] Approach

[0201] To determine the comparative composition of Matriarch, BME and Matrigel as well as the consistency between different preparations of these extracellular matrices, three different batches of matrigel prepared as described in this application were investigated in a LC MS / MS analysis and contrasted to a commercial batch of BME acquired from Cultrex. For further insight the results were compared to two separate lots of the current market golden standard growth factor reduced Matrigel (Al 105 & A3105, CAT# 356234, BD Biosciences, Mississauga, Canada), the latter two by using the data described in Hughes et al. 2010 (" Matrigel: a complex protein mixture required for optimal growth of cell culture". Proteomics. 10 (9): 1886-90; DOI 10.1002 / pmic.200900758). For comparison of composition, a qualitative analysis was done on the relative abundance elective extracellular matrix components, whereas a quantitative analysis was done by integrating the full number of peptide counts hailing from the following categories of ECM components: Collagens (in casu all gene names using the string COL*) ECM Glycoproteins incl. Lamins (in casu all gene names using the strings LAM*, NID*, FGG, PXDN); ECM modifiers (using the strings P4HA*, SERPIN*, HPX, ANXA*, LGALS3*), Proteoglycans (using the string HSPG*) and Others (all peptides mapping back to all remaining peptides. For Matrigel, only gene product for which the total number of peptides retrieved > 10 were included in this comparative analysis, for practical reasons. For assessing batch-to-batch variability, apart from quantitative analysis as to ECM composition, also Pearson coefficients for the entire curated datasets were calculated as well as those for collagens only, an analysis that better captures variability for less high expressed as well.

[0202] LC MS / MS analysis

[0203] Lyophilized samples of three batches of Matriarch and BME were rehydrated in 100-200 pl extraction buffer with 100 mM ammonium bicarbonate +8 M urea. Samples were homogenized using a Bioruptor®Plus (Diagenode SA) at 4 °C for 40 cycles, 15 s ON / OFF. This was followed by a centrifuge step (14,000 g, 15min) and the protein content of supernatants was determined using Pierce BCA Protein Assay Kit (Thermo Scientific). 30 pg of protein was processed further by reduction with 5 mM tris-2-carboxyethyl phosphine for 30 min at 37 °C and subsequently alkylated with 10 mM iodoacetamide for 45 min at RT. This was followed by overnight trypsin digestion at 37 °C. Digestion was stopped by addition of trifluoro acetic acid to a pH lower than 3. Desalting was performed using C18 reversed-phase spin columns (UltraMicro Spin Column, Nest group) according tomanufacturer's instructions. After desalting, samples were resuspended in 2% acetonitrile and 0.1% trifluoroacetic acid. Peptide concentrations were determined using a NanoDrop 2000c (Thermo Scientific).

[0204] Further analysis was performed on Tribrid mass spectrometer (MS) Orbitrap Fusion equipped with a Nanospray source and coupled with an EASY-nLC 1000 ultrahigh pressure liquid chromatography pump (Thermo Fischer Scientific). One microgram of peptide was loaded and concentrated on an Acclaim PepMap 100C18 precolumn (75 pm x 2 cm, Thermo Scientific) and then separated on an Acclaim PepMap RSLC column (75 pm x 25 cm, nanoViper, C18, 2 pm, 100 A) with a column temperature of 45 °C. Peptides were eluted by a nonlinear 2 h gradient at the flow rate of 300 nl / min from 2% solvent B (0.1% formic acid / ACN, Merck) / 98% Solvent A (0.1% formic acid in water, Merck) to 40% solvent B.

[0205] The Orbitrap Fusion was operated in the positive data- dependent acquisition mode. Full MS survey scans from m / z 375-1500 with a resolution 120,000 were performed in the Orbitrap detector. The automatic gain control target was set to 4 x 105 with an injection time of 50 ms. The most intense ions (up to 20) with charge states 2-7 from the full MS scan were selected for fragmentation. MS2 precursors were isolated with a quadrupole mass filter set to a width of 1.2 m / z. Precursors were fragmented by Higher Energy Collision Dissociation and detected in Orbitrap detector with the resolution of 30,000. The normalized collision energy in HCD cell was set 30%. The values for the automatic gain control target and injection time were 5 x 104 and 54 ms, respectively. The duration of dynamic exclusion was set 45s and the mass tolerance window 10 parts per million (PPM).

[0206] Analysis of raw files was performed with MaxQuant (version 2.0.1.0). The resulting peak lists were searched in Andromeda against a reviewed human UniProtKB database (release 2020_04), complemented with the standard MaxQuant contaminant database. Enzyme specificity was set to trypsin / P with a maximum of two missed cleavages. Precursor mass tolerance was set to 4.5 PPM and fragment ion mass tolerance to 20 PPM. Carb amidomethylation of cysteine was used as fixed modification and deamidation (Asparagine), oxidation (Methionine), hydroxyproline and acetylation were considered as variable modifications. The false discovery rate was set to 0.01 for both peptides and proteins, “match between runs” was enabled. Additional data analysis was done on LFQintensities in R (version 4.0.3). EdgeR (version 3.32.1) and Limma (version 3.46.0) R packages were used for differential expression analysis.ICO initiation in BME versus Matriarch

[0207] Viably frozen human liver biopsies were partially thawed until a small ice clump containing tissue could be removed from the cryotubes. Tissue was washed in cold ADV+. Organoid initiation was performed as previously described [Broutier, L., Andersson-Rolf, A., Hindley, C. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. NatProtoc 11, 1724-1743 (2016). https: / / doi.org / 10.1038 / nprot.2016.097]. In short, liver tissue was digested in 2.5 mg / ml Collagenase type A (Sigma) for 20 min at 37 °C. Afterwards, the suspension was strained (70 pm cell strainer) and washed with cold ADV+. The cell pellet was resuspended in reduced growth factor basement membrane matrix (BME, Cultrex) solution or Matriarch solution. The cell suspension was plated in 25 pl droplets in 48-well suspension culture plates (Greiner Bio One). The BME or Matriarch was allowed to solidify for 45-60 min at 37 °C before 250 pl startup expansion medium (SEM) was added. After 72 h, SEM was replaced with expansion medium (EM).

[0208] Intrahepatic cholangiocyte organoids (ICO) were cultured in BME or Matriarch according to previously published protocols (Broutier, L., Andersson-Rolf, A., Hindley, C. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 11, 1724-1743 (2016); DOI: 10.1038 / nprot.2016.097). EM was refreshed every 3-4 days and organoids were typically split every 7-10 days by mechanical dissociation. Organoid fragments were replated in fresh 25 pl BME / Matriarch droplets. Split ratios were empirically determined to maintain the 7-10 day ICO split schedule. Typically, ratios ranged from 1:4 to 1:8. Expansion of organoids was visually qualitatively assessed, both for the capacity to support ICO expansion per se as well as for success rate for ICO expansion from human adult liver biopsies. To assess and functional differentiation contract, in representative cultures of initiated ICOs in BME and Matriarch respectively, undifferentiated stem-cell like epithelium, epithelial bile-duct epithelium and putative liver parenchyma was identified by a skilled experimental hepatologist and the corresponding area was quantified using Imaged and expressed as relative values.

[0209] Transfer from BME to Matriarch hydrogel

[0210] ICO initiated in BME were transferred to LECM hydrogels after they were allowed to proliferate in BME for 2 passages. ICO were harvested by mechanical dissociation. Residual BME was washed away with cold ADV+ and centrifuged (453RCF, 4 °C). The resulting cell pellet was resuspended in cold Matriarch hydrogel. The cell-Matriarch suspensions were plated in 25 pl droplets and wereallowed to solidify at 37 °C for 45-60 min before 250 pil EM was added. Medium was refreshed every 3-4 days. Growth was assessed visually in a qualitative manner.

[0211] Results

[0212] Matriarch, and BME / Matrigel are distinct entities

[0213] A tryptic peptide mapping analysis was performed on a Matrigel-equivalent product (BME). The resulting peptides (91 peptides above the stated count threshold of >4.4 million) confirm a murine core matrisome signature dominated by laminin subunits, nidogen, perlecan, and type IV collagen components. The top 10 proteins (“core matrisome”) reported from this analysis are shown in Table 1.

[0214] Table 1. Top 10 proteins (“core matrisome”) reported for Matrigel-equivalent BME in the provided tryptic peptide mapping dataset.

[0215] Gene Division Category Counts LAMC1 Core matrisome ECM Glycoproteins 53608000000 LAMB1 Core matrisome ECM Glycoproteins 51891000000 LAMA1 Core matrisome ECM Glycoproteins 54278000000 NIDI Core matrisome ECM Glycoproteins 41842000000 HSPG2 Core matrisome Proteoglycans 1411400000 COL4A1 Core matrisome Collagens 947040000 NID2 Core matrisome ECM Glycoproteins 926240000 COL4A2 Core matrisome Collagens 618720000 PXDN Core matrisome ECM Glycoproteins 399320000

[0216] FGG Core matrisome ECM Glycoproteins 256880000

[0217]

[0218] Notably, the BME dataset also highlights the absence of peptides from several fibrillar collagen genes commonly relevant to tissue mechanics and cell-matrix signalling. The provided list of collagen genes not detected in the BME dataset is reproduced in Table 2.

[0219] Table 2. Collagen genes reported as not detected (0 counts) in the provided Matrigel / BME proteomics dataset.

[0220] Gene Division Category Counts COL1A1 Core matrisome Collagens 0

[0221] COL1A2 Core matrisome Collagens 0

[0222] COL3A1 Core matrisome Collagens 0

[0223] COL5A2 Core matrisome Collagens 0

[0224] COL5A1 Core matrisome Collagens 0

[0225] COL6A3 Core matrisome Collagens 0

[0226]

[0227] COL6A2 Core matrisome Collagens 0 COL6A1 Core matrisome Collagens 0

[0228] COL2A1 Core matrisome Collagens 0

[0229] COL5A3 Core matrisome Collagens 0

[0230]

[0231] In a separate mass spectrometry experiment, Matriarch shows substantially greater proteomic diversity. Across three Matriarch batches, 9,738 unique peptides were identified, mapping to 2,129 annotated unique genetic identifiers. Collagen-associated differences are particularly prominent: 252 unique peptides mapped to 23 collagen genes were detected in Matriarch, including strong signals from COL1A2 (~lxlOA12 counts), COL6A2 (~7xlOA8 counts), and additional signals from COL5A1, COL5A2, COL5A3, and COL6A1. Figure 4 shows the analysis of the content of three separate batches of Matriarch, a batch of BME and two batches of Matrigel. Note the collagen dominance of Matriarch over the Matrigel and equivalent products. Also note the widely variant composition of Matrigel and the superior performance of Matriarch in batch-to-batch consistency in this respect. These results indicate that Matriarch and Matrigel / BME are widely divergent ECM extracts with limited compositional resemblance.

[0232] The latter notion is supported by a quantitative analysis based by contrasting the full mass-spec derived contents, again highlighting the important role of collagens over various batches of Matriarch and the important role of Lamins in Matrigel and its equivalent. The latter analysis also indicates high batch-to-batch superiority for Matriarch as compared to Matrigel.

[0233] Batch-to-batch similarity was assessed using Pearson correlation coefficients computed from peptide counts across three Matriarch batches. The correlation matrix based on all 9,783 peptides is shown in Table 3.

[0234] Table 3. Pearson correlation coefficients between Matriarch batches using counts from all detected peptides (n=9,783).

[0235] Batch 1 2 3

[0236] 1 X 0,853353233 0,905302224 2 X X 0,966261758 3 X X X

[0237]

[0238] Because collagen-associated peptides are considered major drivers of matrixdependent proliferation, differentiation and maintenance in many tissue culturecontexts, the same analysis was repeated using only collagen-derived peptides. The resulting correlation matrix is shown in Table 4.

[0239] Table 4. Pearson correlation coefficients between Matriarch batches using counts from collagen-derived peptides.

[0240] Batch 1 2 3

[0241] 1 X 0,990818796 0,983059433 2 X X 0,984846745 3 X X X

[0242]

[0243] Across the three batches, overall peptide correlations range from 0.85 to 0.97, and collagen-peptide correlations range from 0.98 to 0.99, indicating high compositional similarity and supporting improved batch consistency.

[0244] Analytical evidence: Matriarch is consistent with a cow / yak hybrid origin The Matriarch peptide identifications were evaluated for species specificity. Among 9,783 unique peptides detected in the Matriarch mass spectrometry dataset, 611 peptides could be mapped uniquely to Bos taurus, with a similar number mapping uniquely to Bos mutus / Bos grunniens. The remaining peptides mapped to both species and, in many cases, to other members of the genus Bos (e.g., Bos indicus). This mixed mapping pattern is consistent with Matriarch being derived from cow / yak hybrid source tissue.

[0245] Matriarch can replace Matrigel for organoid expansion

[0246] We contrasted Matriarch to the existing standard (BME, Cultrex) for organoid growth. From figure 5 it is clear that both support intrahepatic organoid growth. This effect was seen with three different batches of Matriarch. Despite phenotypic differences gross visual inspection is consistent with both products - despite distinct constituency - being capable of supporting routine growth of organoids. Thus generally speaking, Matriarch can replace Matrigel for routine organoid expansion purposes.

[0247] Initiation from challenging frozen clinical liver biopsies

[0248] Initiating organoids from frozen biopsies is described as highly challenging, particularly when biopsies derive from end-stage liver disease in older transplantrecipient patients. Using a success definition of sustained proliferation for at least five passages, Matriarch achieved a success rate exceeding 70% (accounting for handling / pipetting errors), see Figure 6. This supports non-inferior initiation performance relative to Matrigel under the described conditions.Application historically not well supported by Matrigel is feasible using Matriarch

[0249] A striking observation is that Matriarch is highly superior to Matrigel for creating self-organising branched structures that include polarised cholangiocyte-like epithelium with duct-like (cholangiocyte-like) and parenchyma-like (hepatocyte -like) compartments within the same culture, as demonstrated in Figure 7. It turned out that using Matriarch helps meeting this challenge relatively easy. Upon initiation and subsequent passaging branching structures of apparently cholangiocyte-epithelial identity develop and obtain increasing complexity along with polarised characteristics.

[0250] Gene expression of selected genes revealed that ICO are relative similar between Matrigel® and Matriarch, however, subtle differences have been noted, see Figure 8. For one, expression of Cytokeratin-7 (KRT-7), a cholangiocyte marker, was decreased in Matriarch (2.1-fold lower, *P<0.05). It has been suggested in literature, based on extensive single-cell sequencing of ICO and healthy tissue, that KRT-7 is overexpressed in ICO compared to tissue. These results might indicate that ICO grown in Matriarch reflect a more mature cholangiocyte-like phenotype. No Significant differences were found for other cholangiocytes markers (EPCAM), Organoid markers (LGR-5) and proliferation (KI-67). This indicates that while more mature, the organoids can be expanded without losing important ICO characteristics.

[0251] Initiating organoids from human liver biopsies obtained from explanted livers of transplant recipients is widely regarded as challenging. These specimens derive from end-stage liver disease in typically older patients with severely impaired hepatic function and extensive tissue remodelling. Under these conditions, Matrigel® can achieve modest initiation rates, and the resulting organoids frequently exhibit unidirectional lineage bias: cholangiocyte-like differentiation (with bile-duct-like branching) is fairly commonly observed, whereas hepatocyte -like differentiation is inconsistent and often limited in Matrigel®. In our hands and under standard conditions, Matrigel does not reliably support the concurrent emergence of both cholangiocyte- and hepatocyte -like compartments within a single culture, whereas Matriarch does. However, gene expression of hepatocyte markers Albumin and CYP-3a4 reveal that expression of these genes is slightly upregulated compared to paired organoids grown in Matrigel® (figure 8). This upregulation can be a direct result of the presence of tissue-specific liver ECM components present in the Matriarch and Matriarch can be used to improve current hepatocyte differentiation protocols.Conclusion

[0252] This validation demonstrates that Matriarch can replace Matrigel® for routine human liver organoid expansion and achieves acceptable initiation rates from difficult clinical material (explanted livers from transplant recipients), with a success rate exceeding 70% under our criteria (sustained proliferation for >5 passages). Notably, Matriarch supported concurrent emergence of duct dike (cholangiocyteJike) and parenchymaJike (hepatocyte dike) compartments within the same culture, an application that was not consistently achievable on Matrigel under equivalent conditions. Thus the results underscore that Matriarch Extracellular Hydrogels constitute a unique product, fully different from existing hydrogels and constitutes a rupture with the current state-ofdhe art with respect to growth of liver organoids.

Claims

Claims1. A method for preparing an extracellular matrix (ECM) extract, comprising:- providing organ tissue of a foetal or neonatal animal of the genus Bos, - decellularizing the organ tissue, and- digestion of the decellularized organ tissue.

2. The method according to claim 1, wherein the animal is a foetal or stillborn foetal animal.

3. The method according to claim 1 or 2, comprising in the indicated order:- providing organ tissue of a foetal or neonatal animal of the genus Bos, - decellularizing the organ tissue,- dehydrating the decellularized organ tissue to obtain a powder,- digestion of the powder, and- optionally reconstituting or diluting the mixture obtained in the previous step in an aqueous buffer to obtain a pre-gel solution.

4. The method according to any one of the preceding claims for preparing an ECM based hydrogel, further comprising allowing the decellularized and digested organ tissue, reconstituted powder or pre-gel solution to form a hydrogel.

5. The method according to any one of the preceding claims, wherein the animal is a yak (Bos grunniens or Bos mutus) or a hybrid of a yak and another species of the genus Bos.

6. The method according to any one of the preceding claims, wherein the animal is a hybrid of a yak (Bos grunniens or Bos mutus) and a cow (Bos taurus or Bos indicus).

7. The method according to any one of the preceding claims, wherein the animal is a foetal hybrid of a yak (Bos grunniens or Bos mutus) and a cow (Bos taurus or Bos indicus), preferably a still-born foetus.

8. The method according to any one of the preceding claims, wherein the organ tissue comprises liver tissue, skin tissue, kidney tissue, lung tissue, heart tissue, muscle tissue, stomach tissue, pancreatic tissue, lymph nodes, thymus, tendon tissue, bone tissue, intestine tissue, brain tissue, bladder tissue, genitalorgan tissue, breast tissue, dental tissue, extra-embryonic tissue (including placenta and yolk sac) and combinations thereof.

9. The method according to any one of the preceding claims, wherein the ECM extract is in the form of a hydrogel, a powder or pre-gel solution.

10. The method according to any one of the preceding claims, wherein:- decellularization comprises perfusing the organ tissue with a buffer comprising a surfactant, and treatment with DNase,- dehydration is performed by lyophilization, and / or- allowing the pre- gel solution to form a hydrogel at a temperature of 12°C - 42°C, preferably at about 18°C - 37°C.

11. An extracellular matrix (ECM) extract, hydrogel or derivative thereof obtainable by a method according to any one of claims 1-10.

12. An extracellular matrix (ECM) extract, hydrogel or derivative thereof derived from organ tissue of a foetal or neonatal animal of the genus Bos.

13. The ECM extract, hydrogel or derivative thereof according to claim 12, wherein the animal is a foetal or still-born foetal animal.

14. The ECM extract, hydrogel or derivative thereof according to any one of claims 11-13, comprising yak-derived collagen, yak-derived laminin, and / or yak-derived nidogen-1, preferably wherein the yak is Bos grunniens or Bos mutus.

15. The ECM extract, hydrogel or derivative thereof according to any one of claims 11-14, comprising cow-derived collagen, cow-derived laminin, and / or cow-derived nidogen-1, preferably wherein the cow is Bos taunts or Bos indicus.

16. The ECM extract, hydrogel or derivative thereof according to any one of claims 12-15, wherein the animal is a yak (Bos grunniens or Bos mutus) or a hybrid of a yak and another species of the genus Bos, preferably a hybrid of a yak (Bos grunniens or Bos mutus) and a dairy cow (Bos taunts or Bos indicus), preferably wherein the animal is a foetus, such as a still-born foetus.

17. The ECM extract, hydrogel or derivative thereof according to any one of claims 11-16, comprising 70-95% collagen.

18. Use of an ECM extract, hydrogel or derivative thereof according to any one of claims 11-17 as a substrate for cell, tissue or organoid culturing.

19. A cell, tissue or organoid culture substrate comprising an ECM extract, hydrogel or derivative thereof according to any one of claims 11-17.

20. A method for culturing cells comprising providing an ECM extract, hydrogel or derivative thereof according to any one of claims 11-17 and culturing cells, preferably wherein the cells form a multicellular tissue or organoid.

21. A method or use according to any one of claims 18-20 wherein the ECM extract, hydrogel or derivative thereof supports the maintenance, growth and differentiation of organoids.

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