Organoids in skin wound healing

Abstract

Stem cells (SCs) can self-replicate and differentiate into multiple lineages. Organoids, 3D cultures derived from SCs, can replicate the spatial structure and physiological characteristics of organs in vitro. Skin organoids can effectively simulate the physiological structure and function of skin tissue, reliably restoring the natural skin ecology in various in vitro environments. Skin organoids have been employed extensively in skin development and pathology research, offering valuable insights for drug screening. Moreover, they play crucial roles in skin regeneration and tissue repair. This in-depth review explores the construction and applications of skin organoids in wound healing, with a focus on their construction process, including skin appendage integration, and significant advancements in wound-healing research.

Keywords

Skin organoids / Tissue engineering / Pluripotent stem cells / Skin wound healing

References

[1] Bacakova L, Zarubova J, Travnickova M, Musilkova J, Pajorova J, Slepicka P, et al. Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells-a review. Biotechnol Adv. 2018; 36: 1111-26. https://doi.org/10.1016/j.biotechadv.2018.03.011.

[2] Siminovitch L, Mcculloch EA, Till JE. The distribution of colony-forming cells among spleen colonies. J Cell Comp Physiol. 1963; 62: 327-36. https://doi.org/10.1002/jcp.1030620313.

[3] Yamanaka S.Pluripotent stem cell-based cell therapy-promise and challenges. Cell Stem Cell. 2020; 27: 523-31. https://doi.org/10.1016/j.stem.2020.09.014.

[4] Jin J. Stem cell treatments. Jama-J Am Med Assoc. 2017; 317: 330. https://doi.org/10.1001/jama.2016.17822.

[5] Dutta D, Heo I, Clevers H. Disease Modeling in stem cell-derived 3D organoid systems. Trends Mol Med. 2017; 23: 393-410. https://doi.org/10.1016/j.molmed.2017.02.007.

[6] Duval K, Grover H, Han LH, et al. Modeling physiological events in 2D vs. 3D cell culture. Physiology. 2017; 32: 266-77. https://doi.org/10.1152/physiol.00036.2016.

[7] Jensen C, Teng Y. Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci. 2020; 7: 33. https://doi.org/10.3389/fmolb.2020.00033.

[8] Hosseini M, Koehler KR, Shafiee A. Biofabrication of human skin with its appendages. Adv Healthc Mater. 2022; 11: e2201626.

[9] Velasco S, Kedaigle AJ, Simmons SK, et al. Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature. 2019; 570: 523-7. https://doi.org/10.1038/s41586-019-1289-x.

[10] Wu H, Uchimura K, Donnelly EL, et al. Comparative analysis and refinement of human PSC-derived kidney organoid differentiation with single-cell transcriptomics. Cell Stem Cell. 2018; 23: 869-881.e8. https://doi.org/10.1016/j.stem.2018.10.010.

[11] Seidlitz T, Merker SR, Rothe A, et al. Human gastric cancer modelling using organoids. Gut. 2019; 68: 207-17. https://doi.org/10.1136/gutjnl-2017-314549.

[12] Sachs N, de Ligt J, Kopper O, et al. A living biobank of breast cancer organoids captures disease heterogeneity. Cell. 2018; 172: 373-386.e10. https://doi.org/10.1016/j.cell.2017.11.010.

[13] Lim K, Donovan APA, Tang W, et al. Organoid modeling of human fetal lung alveolar development reveals mechanisms of cell fate patterning and neonatal respiratory disease. Cell Stem Cell. 2023; 30: 20-37.e9. https://doi.org/10.1016/j.stem.2022.11.013.

[14] Shi X, Li Y, Yuan Q, et al. Integrated profiling of human pancreatic cancer organoids reveals chromatin accessibility features associated with drug sensitivity. Nat Commun. 2022; 13: 2169. https://doi.org/10.1038/s41467-022-29857-6.

[15] Schutgens F, Clevers H. Human organoids: tools for understanding biology and treating diseases. Annu Rev Pathol-Mech Dis. 2020; 15: 211-34. https://doi.org/10.1146/annurev-pathmechdis-012419-032611.

[16] Abdullah KG, Bird CE, Buehler JD, et al. Establishment of patient-derived organoid models of lower-grade glioma. Neuro-Oncology. 2022; 24: 612-23. https://doi.org/10.1093/neuonc/noab273.

[17] Kageyama T, Miyata H, Seo J, et al. In vitro hair follicle growth model for drug testing. Sci Rep. 2023; 13: 4847. https://doi.org/10.1038/s41598-023-31842-y.

[18] Sampaziotis F, Muraro D, Tysoe OC, et al. Cholangiocyte organoids can repair bile ducts after transplantation in the human liver. Science. 2021; 371: 839-46. https://doi.org/10.1126/science.aaz6964.

[19] Gravitz L. Skin. Nature. 2018; 563: S83. https://doi.org/10.1038/d41586-018-07428-4.

[20] Peña OA, Martin P. Cellular and molecular mechanisms of skin wound healing. Nat Rev Mol Cell Biol. 2024; 25: 599-616. https://doi.org/10.1038/s41580-024-00715-1.

[21] Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975; 6: 331-43. https://doi.org/10.1016/S0092-8674(75)80001-8.

[22] Lee J, Koehler KR. Skin organoids: a new human model for developmental and translational research. Exp Dermatol. 2021; 30: 613-20. https://doi.org/10.1111/exd.14292.

[23] Zhou S, Li Z, Li X, et al. Crosstalk between endothelial cells and dermal papilla entails hair regeneration and angiogenesis during aging. J Adv Res. 2024;May 7:S2090-1232(24)00183-8. https://doi.org/10.2991/978-94-6463-514-0.

[24] Xiao X, Gao Y, Yan L, et al. M1 polarization of macrophages promotes stress-induced hair loss via interleukin-18 and interleukin-1β. J Cell Physiol. 2024; 239: e31181. https://doi.org/10.1002/jcp.31181.

[25] Ebner-Peking P, Krisch L, Wolf M, et al. Self-assembly of differentiated progenitor cells facilitates spheroid human skin organoid formation and planar skin regeneration. Theranostics. 2021; 11: 8430-47. https://doi.org/10.7150/thno.59661.

[26] Boeckmann L, Berner J, Kordt M, et al. Synergistic effect of cold gas plasma and experimental drug exposure exhibits skin cancer toxicity in vitro and in vivo. J Adv Res. 2024; 57: 181-96. https://doi.org/10.1016/j.jare.2023.06.014.

[27] Sun Y, Revach OY, Anderson S, et al. Targeting TBK1 to overcome resistance to cancer immunotherapy. Nature. 2023; 615: 158-67. https://doi.org/10.1038/s41586-023-05704-6.

[28] Wang M, Zhou X, Zhou S, et al. Mechanical force drives the initial mesenchymal-epithelial interaction during skin organoid development. Theranostics. 2023; 13: 2930-45. https://doi.org/10.7150/thno.83217.

[29] Lee J, Böscke R, Tang PC, et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 2018; 22: 242-54. https://doi.org/10.1016/j.celrep.2017.12.007.

[30] Jung SY, You HJ, Kim MJ, et al. Wnt-activating human skin organoid model of atopic dermatitis induced by Staphylococcus aureus and its protective effects by Cutibacterium acnes. Iscience. 2022; 25: 105150-70. https://doi.org/10.1016/j.isci.2022.105150.

[31] Ma J, Liu J, Gao D, et al. Establishment of human pluripotent stem cell-derived skin organoids enabled pathophysiological model of SARS-CoV-2 infection. Adv Sci. 2022; 9: e2104192.

[32] Li P, Pachis ST, Xu G, Schraauwen R, Incitti R, de Vries AC, et al. Mpox virus infection and drug treatment modelled in human skin organoids. Nat Microbiol. 2023; 8: 2067-79. https://doi.org/10.1038/s41564-023-01489-6.

[33] Schörghofer D, Vock L, Mirea MA, Eckel O, Gschwendtner A, Neesen J, et al. Late stage melanoma is hallmarked by low NLGN4X expression leading to HIF1A accumulation. Br J Cancer. 2024; 131: 468-80. https://doi.org/10.1038/s41416-024-02758-9.

[34] Hong ZX, Zhu ST, Li H, Luo JZ, Yang Y, An Y, et al. Bioengineered skin organoids: from development to applications. Military Med Res. 2023; 10: 40. https://doi.org/10.1186/s40779-023-00475-7.

[35] Fu XB. Repair cell first, then regenerate the tissues and organs. Military Med Res. 2021; 8: 2. https://doi.org/10.1186/s40779-021-00297-5.

[36] Yin X, Mead BE, Safaee H, Langer R, Karp JM, Levy O. Engineering stem cell organoids. Cell Stem Cell. 2016; 18: 25-38. https://doi.org/10.1016/j.stem.2015.12.005.

[37] Boonekamp KE, Kretzschmar K, Wiener DJ, Asra P, Derakhshan S, Puschhof J, et al. Long-term expansion and differentiation of adult murine epidermal stem cells in 3D organoid cultures. Proc Natl Acad Sci USA. 2019; 116: 14630-8. https://doi.org/10.1073/pnas.1715272116.

[38] Diao J, Liu J, Wang S, Chang M, Wang X, Guo B, et al. Sweat gland organoids contribute to cutaneous wound healing and sweat gland regeneration. Cell Death Dis. 2019; 10: 238. https://doi.org/10.1038/s41419-019-1485-5.

[39] Jiang J, Shao X, Liu W, Wang M, Li Q, Wang M, et al. The mechano-chemical circuit in fibroblasts and dendritic cells drives basal cell proliferation in psoriasis. Cell Rep. 2024; 43: 114513. https://doi.org/10.1016/j.celrep.2024.114513.

[40] Perez WB, Cable CJ, Shi B, Ventrella R, Kaplan N, Kobeissi A, et al. Receptor tyrosine kinase EPHA2 drives epidermal differentiation through regulation of EGFR Signaling. J Invest Dermatol. 2024; 144: 1798-1807.e1. https://doi.org/10.1016/j.jid.2024.01.014.

[41] Jo H, Brito S, Kwak BM, Park S, Lee MG, Bin BH. Applications of mesenchymal stem cells in skin regeneration and rejuvenation. Int J Mol Sci. 2021; 22: 2410-29. https://doi.org/10.3390/ijms22052410.

[42] Su Y, Wen J, Zhu J, Xie Z, Liu C, Ma C, et al. Pre-aggregation of scalp progenitor dermal and epidermal stem cells activates the WNT pathway and promotes hair follicle formation in in vitro and in vivo systems. Stem Cell Res Ther. 2019; 10: 403. https://doi.org/10.1186/s13287-019-1504-6.

[43] Li KN, Tumbar T. Hair follicle stem cells as a skin-organizing signaling center during adult homeostasis. EMBO J. 2021; 40: e107135-54. https://doi.org/10.15252/embj.2020107135.

[44] Chen P, Miao Y, Zhang F, Huang J, Chen Y, Fan Z, et al. Nanoscale microenvironment engineering based on layer-by-layer self-assembly to regulate hair follicle stem cell fate for regenerative medicine. Theranostics. 2020; 10: 11673-89. https://doi.org/10.7150/thno.48723.

[45] Koehler KR, Mikosz AM, Molosh AI, Patel D, Hashino E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature. 2013; 500: 217-21. https://doi.org/10.1038/nature12298.

[46] Lee J, Rabbani CC, Gao H, Steinhart MR, Woodruff BM, Pflum ZE, et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature. 2020; 582: 399-404. https://doi.org/10.1038/s41586-020-2352-3.

[47] Yilmaz A, Braverman-Gross C, Bialer-Tsypin A, Peretz M, Benvenisty N. Mapping gene circuits essential for germ layer differentiation via loss-of-function screens in haploid human embryonic stem cells. Cell Stem Cell. 2020; 27: 679-691.e6. https://doi.org/10.1016/j.stem.2020.06.023.

[48] Jamwal VS, Vishnu VV, Domreddy A, Parekh Y, Kumar BK, Chandra Shekar P, et al. Generation of iPSC from fetal fibroblast cells obtained from an abortus with type-I tri-allelic variants. Stem Cell Res. 2020; 48: 101963. https://doi.org/10.1016/j.scr.2020.101963.

[49] Kim Y, Park N, Rim YA, Nam Y, Jung H, Lee K, et al. Establishment of a complex skin structure via layered co-culture of keratinocytes and fibroblasts derived from induced pluripotent stem cells. Stem Cell Res Ther. 2018; 9: 217. https://doi.org/10.1186/s13287-018-0958-2.

[50] Yang YH, Zhang RZ, Cheng S, Xu B, Tian T, Shi HX, et al. Generation of induced pluripotent stem cells from human epidermal keratinocytes. Cell Reprogramm. 2018; 20: 356-64. https://doi.org/10.1089/cell.2018.0035.

[51] Kim Y, Ju JH. Generation of 3D skin organoid from cord blood-derived induced pluripotent stem cells. J Vis Exp. 2019; 146: e59297-306. https://doi.org/10.3791/59297.

[52] Sah SK, Kanaujiya JK, Chen IP, Reichenberger EJ.Generation of keratinocytes from human induced pluripotent stem cells under defined culture conditions. Cell Reprogramm. 2021; 23: 1-13. https://doi.org/10.1089/cell.2020.0046.

[53] Itoh M, Kiuru M, Cairo MS, Christiano AM. Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells. Proc Natl Acad Sci USA. 2011; 108: 8797-802. https://doi.org/10.1073/pnas.1100332108.

[54] Shafiee A, Sun J, Ahmed IA, Phua F, Rossi GR, Lin CY, et al. Development of physiologically relevant skin organoids from human induced pluripotent stem cells. Small. 2024; 20:e2304879.

[55] Kim S, Min S, Choi YS, Jo SH, Jung JH, Han K, et al. Tissue extracellular matrix hydrogels as alternatives to Matrigel for culturing gastrointestinal organoids. Nat Commun. 2022; 13: 1692. https://doi.org/10.1038/s41467-022-29279-4.

[56] Li T, Hou J, Wang L, Zeng G, Wang Z, Yu L, et al. Bioprinted anisotropic scaffolds with fast stress relaxation bioink for engineering 3D skeletal muscle and repairing volumetric muscle loss. Acta Biomater. 2023; 156: 21-36. https://doi.org/10.1016/j.actbio.2022.08.037.

[57] Sun Y, You Y, Wu Q, Hu R, Dai K. Senescence-targeted MicroRNA/organoid composite hydrogel repair cartilage defect and prevention joint degeneration via improved chondrocyte homeostasis. Bioact Mater. 2024; 39: 427-42. https://doi.org/10.1016/j.bioactmat.2024.05.036.

[58] Gan Z, Qin X, Liu H, Liu J, Qin J. Recent advances in defined hydrogels in organoid research. Bioact Mater. 2023; 28: 386-401. https://doi.org/10.1016/j.bioactmat.2023.06.004.

[59] Shen C, Wang J, Li G, Hao S, Wu Y, Song P, et al. Boosting cartilage repair with silk fibroin-DNA hydrogel-based cartilage organoid precursor. Bioact Mater. 2024; 35: 429-44. https://doi.org/10.1016/j.bioactmat.2024.02.016.

[60] Carpentier N, Ye S, Delemarre MD, van der Meeren L, Skirtach AG, van der Laan LJW, et al. Gelatin-based hybrid hydrogels as matrices for organoid culture. Biomacromolecules. 2024; 25: 590-604. https://doi.org/10.1021/acs.biomac.2c01496.

[61] Quílez C, Jeon EY, Pappalardo A, Pathak P, Abaci HE. Efficient generation of skin organoids from pluripotent cells via defined extracellular matrix cues and morphogen gradients in a spindle-shaped microfluidic device. Adv Healthc Mater. 2024; 13: e2400405-20. https://doi.org/10.1002/adhm.202400405.

[62] Hosseini M, Shafiee A. Engineering bioactive scaffolds for skin regeneration. Small. 2021; 17: e2101384.

[63] Xie X, Tong X, Li Z, Cheng Q, Wang X, Long Y, et al. Use of mouse primary epidermal organoids for USA300 infection modeling and drug screening. Cell Death Dis. 2023; 14: 15. https://doi.org/10.1038/s41419-022-05525-x.

[64] Wiener DJ, Studer IC, MAT B, Hermann A, Vincenti S, Zhang M, et al. Characterization of canine epidermal organoid cultures by immunohistochemical analysis and quantitative PCR. Vet Dermatol. 2021; 32: 144-79.

[65] Wang X, Wang S, Guo B, Su Y, Tan Z, Chang M, et al. Human primary epidermal organoids enable modeling of dermatophyte infections. Cell Death Dis. 2021; 12: 35. https://doi.org/10.1038/s41419-020-03330-y.

[66] Kwak S, Song CL, Lee J, Kim S, Nam S, Park YJ, et al. Development of pluripotent stem cell-derived epidermal organoids that generate effective extracellular vesicles in skin regeneration. Biomaterials. 2024; 307: 122522. https://doi.org/10.1016/j.biomaterials.2024.122522.

[67] Gupta AC, Chawla S, Hegde A, Singh D, Bandyopadhyay B, Lakshmanan CC, et al. Establishment of an in vitro organoid model of dermal papilla of human hair follicle. J Cell Physiol. 2018; 233: 9015-30. https://doi.org/10.1002/jcp.26853.

[68] Ramovs V, Janssen H, Fuentes I, Pitaval A, Rachidi W, Chuva de Sousa Lopes SM, et al. Characterization of the epidermal-dermal junction in hiPSC-derived skin organoids. Stem Cell Rep. 2022; 17: 1279-88. https://doi.org/10.1016/j.stemcr.2022.04.008.

[69] Weber EL, Woolley TE, Yeh CY, Ou KL, Maini PK, Chuong CM. Self-organizing hair peg-like structures from dissociated skin progenitor cells: new insights for human hair follicle organoid engineering and Turing patterning in an asymmetric morphogenetic field. Exp Dermatol. 2019; 28: 355-66. https://doi.org/10.1111/exd.13891.

[70] Veraitch O, Kobayashi T, Imaizumi Y, Akamatsu W, Sasaki T, Yamanaka S, et al. Human induced pluripotent stem cell-derived ectodermal precursor cells contribute to hair follicle morphogenesis in vivo. J Invest Dermatol. 2013; 133: 1479-88. https://doi.org/10.1038/jid.2013.7.

[71] Marinho PA, Jeong G, Shin SH, Kim SN, Choi H, Lee SH, et al. The development of an in vitro human hair follicle organoid with a complexity similar to that in vivo. Biomed Mater. 2024; 19: 025041. https://doi.org/10.1088/1748-605X/ad2707.

[72] Kim MJ, Ahn HJ, Kong D, Lee S, Kim DH, Kang KS. Modeling of solar UV-induced photodamage on the hair follicles in human skin organoids. J Tissue Eng. 2024; 15: 1788739057.

[73] Feldman A, Mukha D, Maor II, Sedov E, Koren E, Yosefzon Y, et al. Blimp1(+) cells generate functional mouse sebaceous gland organoids in vitro. Nat Commun. 2019; 10: 2348. https://doi.org/10.1038/s41467-019-10261-6.

[74] Wang X, Wang X, Liu J, Cai T, Guo L, Wang S, et al. Hair follicle and sebaceous gland De novo regeneration with cultured epidermal stem cells and skin-derived precursors. Stem Cells Transl Med. 2016; 5: 1695-706. https://doi.org/10.5966/sctm.2015-0397.

[75] Zhang C, Chen Y, Fu X. Sweat gland regeneration after burn injury: is stem cell therapy a new hope? Cytotherapy. 2015; 17: 526-35. https://doi.org/10.1016/j.jcyt.2014.10.016.

[76] Chen R, Zhu Z, Ji S, Geng Z, Hou Q, Sun X, et al. Sweat gland regeneration: current strategies and future opportunities. Biomaterials. 2020; 255: 120201-20. https://doi.org/10.1016/j.biomaterials.2020.120201.

[77] Sun X, Xiang J, Chen R, Geng Z, Wang L, Liu Y, et al. Sweat gland organoids originating from reprogrammed epidermal keratinocytes functionally recapitulated damaged skin. Adv Sci. 2021; 8: e2103079.

[78] Yuan X, Duan X, Enhejirigala, Li Z, Yao B, Song W, et al. Reciprocal interaction between vascular niche and sweat gland promotes sweat gland regeneration. Bioact. Mater. 2023; 21: 340-57. https://doi.org/10.1016/j.bioactmat.2022.08.021.

[79] Sen CK. Human wounds and its burden: An updated compendium of estimates. Adv Wound Care. 2019; 8: 39-48. https://doi.org/10.1089/wound.2019.0946.

[80] Takagi R, Ishimaru J, Sugawara A, Toyoshima KE, Ishida K, Ogawa M, et al. Bioengineering a 3D integumentary organ system from iPS cells using an in vivo transplantation model. Sci Adv. 2016; 2: e1500887. https://doi.org/10.1126/sciadv.1500887.

[81] Ma J, Li W, Cao R, Gao D, Zhang Q, Li X, et al. Application of an iPSC-derived organoid model for localized scleroderma therapy. Adv Sci. 2022; 9: e2106075.

[82] Thai VL, Ramos-Rodriguez DH, Mesfin M, Leach JK. Hydrogel degradation promotes angiogenic and regenerative potential of cell spheroids for wound healing. Mater Today Bio. 2023; 22: 100769. https://doi.org/10.1016/j.mtbio.2023.100769.

[83] Choudhury S, Dhoke NR, Chawla S, Das A. Bioengineered MSC(Cxcr2) transdifferentiated keratinocyte-like cell-derived organoid potentiates skin regeneration through ERK1/2 and STAT3 signaling in diabetic wound. Cell Mol Life Sci. 2024; 81: 172. https://doi.org/10.1007/s00018-023-05057-3.

[84] Cubo N, Garcia M, Del CJ, Velasco D, Jorcano JL. 3D bioprinting of functional human skin: production and in vivo analysis. Biofabrication. 2016; 9: 15006. https://doi.org/10.1088/1758-5090/9/1/015006.

[85] Jorgensen AM, Gorkun A, Mahajan N, Willson K, Clouse C, Jeong CG, et al. Multicellular bioprinted skin facilitates human-like skin architecture in vivo. Sci Transl Med. 2023; 15: f7547.

[86] Pappalardo A, Alvarez Cespedes D, Fang S, Herschman AR, Jeon EY, Myers KM, et al. Engineering edgeless human skin with enhanced biomechanical properties. Sci Adv. 2023; 9: e2514.

[87] Abaci HE, Coffman A, Doucet Y, Chen J, Jacków J, Wang E, et al. Tissue engineering of human hair follicles using a biomimetic developmental approach. Nat Commun. 2018; 9: 5301. https://doi.org/10.1038/s41467-018-07579-y.

[88] Michael S, Sorg H, Peck CT, Koch L, Deiwick A, Chichkov B, et al. Tissue engineered skin substitutes created by laser-assisted bioprinting form skin-like structures in the dorsal skin fold chamber in mice. PLoS One. 2013; 8: e57741. https://doi.org/10.1371/journal.pone.0057741.

[89] Zhou F, Hong Y, Liang R, Zhang X, Liao Y, Jiang D, et al. Rapid printing of bio-inspired 3D tissue constructs for skin regeneration. Biomaterials. 2020; 258: 120287. https://doi.org/10.1016/j.biomaterials.2020.120287.

[90] Huang S, Yao B, Xie J, Fu X. 3D bioprinted extracellular matrix mimics facilitate directed differentiation of epithelial progenitors for sweat gland regeneration. Acta Biomater. 2016; 32: 170-7. https://doi.org/10.1016/j.actbio.2015.12.039.

[91] Yao B, Wang R, Wang Y, Zhang Y, Hu T, Song W, et al. Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration. Sci Adv. 2020; 6: z1094.

[92] Kang D, Liu Z, Qian C, Huang J, Zhou Y, Mao X, et al. 3D bioprinting of a gelatin-alginate hydrogel for tissue-engineered hair follicle regeneration. Acta Biomater. 2023; 165: 19-30. https://doi.org/10.1016/j.actbio.2022.03.011.

[93] Hartmann-Fritsch F, Biedermann T, Braziulis E, Luginbühl J, Pontiggia L, Böttcher-Haberzeth S, et al. Collagen hydrogels strengthened by biodegradable meshes are a basis for dermo-epidermal skin grafts intended to reconstitute human skin in a one-step surgical intervention. J Tissue Eng Regen Med. 2016; 10: 81-91. https://doi.org/10.1002/term.1665.

[94] Zhao M, Wang J, Zhang J, Huang J, Luo L, Yang Y, et al. Functionalizing multi-component bioink with platelet-rich plasma for customized in-situ bilayer bioprinting for wound healing. Mater Today Bio. 2022; 16: 100334. https://doi.org/10.1016/j.mtbio.2022.100334.

[95] Bacakova M, Pajorova J, Broz A, Hadraba D, Lopot F, Zavadakova A, et al. A two-layer skin construct consisting of a collagen hydrogel reinforced by a fibrin-coated polylactide nanofibrous membrane. Int J Nanomedicine. 2019; 14: 5033-50. https://doi.org/10.2147/IJN.S200782.

[96] Guo Y, Bian Z, Xu Q, Wen X, Kang J, Lin S, et al. Novel tissue-engineered skin equivalent from recombinant human collagen hydrogel and fibroblasts facilitated full-thickness skin defect repair in a mouse model. Mater Sci Eng C-Mater Biol Appl. 2021; 130: 112469. https://doi.org/10.1016/j.msec.2021.112469.

[97] Li X, Xie R, Luo Y, Shi R, Ling Y, Zhao X, et al. Cooperation of TGF-β and FGF signalling pathways in skin development. Cell Prolif. 2023; 56: e13489. https://doi.org/10.1111/cpr.13489.

[98] Xiong S, Zhang X, Lu P, Wu Y, Wang Q, Sun H, et al. A Gelatin-sulfonated silk composite scaffold based on 3D printing technology enhances skin regeneration by stimulating epidermal growth and dermal neovascularization. Sci Rep. 2017; 7: 4288. https://doi.org/10.1038/s41598-017-04149-y.

[99] Hahn S, Kim MS, Choi SY, Jeong S, Jee JH, Kim HK, et al. Leucine-rich repeat-containing G-protein coupled receptor 5 enriched organoids under chemically-defined growth conditions. Biochem Biophys Res Commun. 2019; 508: 430-9. https://doi.org/10.1016/j.bbrc.2018.11.003.

[100] Kageyama T, Seo J, Yan L, Fukuda J. Effects of oxytocin on the hair growth ability of dermal papilla cells. Sci Rep. 2023; 13: 15587. https://doi.org/10.1038/s41598-023-40521-x.

[101] Cruz-Acuña R, Quirós M, Farkas AE, Dedhia PH, Huang S, Siuda D, et al. Synthetic hydrogels for human intestinal organoid generation and colonic wound repair. Nat Cell Biol. 2017; 19: 1326-35.

[102] Marti-Figueroa CR, Ashton RS. The case for applying tissue engineering methodologies to instruct human organoid morphogenesis. Acta Biomater. 2017; 54: 35-44. https://doi.org/10.1016/j.actbio.2017.03.023.

[103] Kopper O, de Witte CJ, Lõhmussaar K, Valle-Inclan JE, Hami N, Kester L, et al. An organoid platform for ovarian cancer captures intra-and interpatient heterogeneity. Nat Med. 2019; 25: 838-49. https://doi.org/10.1038/s41591-019-0422-6.

[104] Gjorevski N, Nikolaev M, Brown TE, Mitrofanova O, Brandenberg N, DelRio FW, et al. Tissue geometry drives deterministic organoid patterning. Science. 2022; 375: w9021.

[105] Chen Y, Tandon I, Heelan W, Wang Y, Tang W, Hu Q. Proteolysis-targeting chimera (PROTAC) delivery system: advancing protein degraders towards clinical translation. Chem Soc Rev. 2022; 51: 5330-50. https://doi.org/10.1039/D1CS00762A.

[106] Veith AP, Henderson K, Spencer A, Sligar AD, Baker AB. Therapeutic strategies for enhancing angiogenesis in wound healing. Adv Drug Deliv Rev. 2019; 146: 97-125. https://doi.org/10.1016/j.addr.2018.09.010.

[107] Salewskij K, Penninger JM. Blood vessel organoids for development and disease. CircRes. 2023; 132: 498-510. https://doi.org/10.1161/CIRCRESAHA.122.321768.

[108] Park JW, Hwang SR, Yoon IS.Advanced growth factor delivery Systems in Wound Management and Skin Regeneration. Molecules. 2017; 22: 1259-79. https://doi.org/10.3390/molecules22081259.

[109] Mori N, Morimoto Y, Takeuchi S. Skin integrated with perfusable vascular channels on a chip. Biomaterials. 2017; 116: 48-56. https://doi.org/10.1016/j.biomaterials.2016.11.031.

[110] Novak R, Ingram M, Marquez S, das D, Delahanty A, Herland A, et al. Robotic fluidic coupling and interrogation of multiple vascularized organ chips. Nat Biomed Eng. 2020; 4: 407-20. https://doi.org/10.1038/s41551-019-0497-x.

[111] Quintard C, Tubbs E, Jonsson G, Jiao J, Wang J, Werschler N, et al. A microfluidic platform integrating functional vascularized organoids-on-chip. Nat Commun. 2024; 15: 1452. https://doi.org/10.1038/s41467-024-45710-4.

[112] Li Q, Niu K, Wang D, Xuan L, Wang X. Low-cost rapid prototyping and assembly of an open microfluidic device for a 3D vascularized organ-on-a-chip. Lab Chip. 2022; 22: 2682-94. https://doi.org/10.1039/D1LC00767J.

[113] Bouffi C, Wikenheiser-Brokamp KA, Chaturvedi P, Sundaram N, Goddard GR, Wunderlich M, et al. In vivo development of immune tissue in human intestinal organoids transplanted into humanized mice. Nat Biotechnol. 2023; 41: 824-31. https://doi.org/10.1038/s41587-022-01558-x.

[114] Múnera JO, Kechele DO, Bouffi C, Qu N, Jing R, Maity P, et al. Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon. Cell Stem Cell. 2023; 30: 1434-1451.e9. https://doi.org/10.1016/j.stem.2023.10.002.

[115] Lovell-Badge R, Anthony E, Barker RA, Bubela T, Brivanlou AH, Carpenter M, et al. ISSCR guidelines for stem cell research and clinical translation: the 2021 update. Stem Cell Rep. 2021; 16: 1398-408. https://doi.org/10.1016/j.stemcr.2021.05.012.

[116] Artegiani B, Hendriks D, Beumer J, Kok R, Zheng X, Joore I, et al. Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing. Nat Cell Biol. 2020; 22: 321-31. https://doi.org/10.1038/s41556-020-0472-5.

[117] Dekkers JF, Whittle JR, Vaillant F, Chen HR, Dawson C, Liu K, et al. Modeling breast cancer using CRISPR-Cas9-mediated engineering of human breast organoids. Jnci-J Natl Cancer Inst. 2020; 112: 540-4. https://doi.org/10.1093/jnci/djz196.

[118] Michels BE, Mosa MH, Streibl BI, Zhan T, Menche C, Abou-el-Ardat K, et al. Pooled In vitro and In vivo CRISPR-Cas9 screening identifies tumor suppressors in human colon organoids. Cell Stem Cell. 2020; 26: 782-792.e7. https://doi.org/10.1016/j.stem.2020.04.003.

[119] Mircetic J, Camgöz A, Abohawya M, Ding L, Dietzel J, Tobar SG, et al. CRISPR/Cas9 screen in gastric cancer patient-derived organoids reveals KDM1A-NDRG1 Axis as a targetable vulnerability. Small Methods. 2023;7:e2201605.

[120] Dabelsteen S, Pallesen EMH, Marinova IN, Nielsen MI, Adamopoulou M, Rømer TB, et al. Essential functions of Glycans in human epithelia dissected by a CRISPR-Cas9-engineered human Organotypic skin model. Dev Cell. 2020; 54: 669-684.e7. https://doi.org/10.1016/j.devcel.2020.06.039.

[121] Gabbin B, Meraviglia V, Angenent ML, Ward-van Oostwaard D, Sol W, Mummery CL, et al. Heart and kidney organoids maintain organ-specific function in a microfluidic system. Mater Today Bio. 2023; 23: 100818. https://doi.org/10.1016/j.mtbio.2023.100818.

[122] Maramraju S, Kowalczewski A, Kaza A, Liu X, Singaraju JP, Albert MV, et al. AI-organoid integrated systems for biomedical studies and applications. Bioeng Transl Med. 2024; 9: e10641. https://doi.org/10.1002/btm2.10641.

[123] Bai L, Wu Y, Li G, Zhang W, Zhang H, Su J. AI-enabled organoids: construction, analysis, and application. Bioact. Mater. 2024; 31: 525-48. https://doi.org/10.1016/j.bioactmat.2023.09.005.

[124] Norkin M, Ordóñez-Morán P, Huelsken J. High-content, targeted RNA-seq screening in organoids for drug discovery in colorectal cancer. Cell Rep. 2021; 35: 109026. https://doi.org/10.1016/j.celrep.2021.109026.

[125] Ji S, Feng L, Fu Z, Wu G, Wu Y, Lin Y, et al. Pharmacoproteogenomic characterization of liver cancer organoids for precision oncology. Sci Transl Med. 2023; 15: g3358.

[126] Farin HF, Mosa MH, Ndreshkjana B, Grebbin BM, Ritter B, Menche C, et al. Colorectal cancer organoid-stroma biobank allows subtype-specific assessment of individualized therapy responses. Cancer Discov. 2023; 13: 2192-211. https://doi.org/10.1158/2159-8290.CD-23-0050.