Name: Forfatter i søkelyset: Fremme senevevsteknikk med 3D-organoidmodeller
Uploaded: 2024-06-21T13:00:00
Description: Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

D.D. og S.M.-D. "CellWiTaL: Reproduserbare cellesystemer for legemiddelforskning - overføring av lagfri laserutskrift av svært spesifikke enkeltceller i tredimensjonale cellulære strukturer" Forslag nr. 13N15874. D.D. og V.R.A. anerkjenner EU MSCA-COFUND Grant OSTASKILLS "Holistisk opplæring av neste generasjons slitasjegiktforskning" GA Nr. 101034412. Alle forfattere anerkjenner fru Beate Geyer for teknisk assistanse.

MERK: Alle trinnene må utføres ved hjelp av aseptiske teknikker.
1. Kultur og pre-utvidelse av NHDFs
<ol><li>Tine raskt kryohetteglasset som inneholder voksne kryopreserverte normale humane fibroblaster (NHDF, 1 x 106 celler) ved 37 °C til de nesten tiner.</li>
	<li>Tilsett sakte 1 ml forvarmet fibroblastvekstmedium 2 (bruksklart sett inkludert basalmedium, 2% føtalkalveserum (FCS), basisk fibroblastvekstfaktor (bFGF) og insulin) supplert med 1% penicillin / s...

Tre-trinns organoidmodell for seneforskning og ingeniørfag

<ol>
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G., Foolen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+injury+and+repair+-+A+perspective+on+the+basic+mechanisms+of+tendon+disease+and+future+clinical+therapy.">Tendon injury and repair - A perspective on the basic mechanisms of tendon disease and future clinical therapy.</a> Acta Biomater. 63, 18-36 (2017).</li><li>Lomas, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+in+scaffold-based+tendon+treatments.">The past, present and future in scaffold-based tendon treatments.</a> Adv Drug Deliv Rev. 84, 257-277 (2015).</li><li>Gomez-Florit, M., Labrador-Rached, C. J., Domingues, R. M. A., Gomes, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tendon+microenvironment:+Engineered+in+vitro+models+to+study+cellular+crosstalk.">The tendon microenvironment: Engineered in vitro models to study cellular crosstalk.</a> Adv Drug Deliv Rev. 185, 114299 (2022).</li><li>Docheva, D., M&#252;ller, S. A., Majewski, M., Evans, C. 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G., Alt, V., Morsczeck, C., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+follicle+cell+differentiation+towards+periodontal+ligament-like+tissue+in+a+self-assembly+three-dimensional+organoid+model.">Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model.</a> Eur Cell Mater. 42, 20-33 (2021).</li><li>Yan, Z., Yin, H., Brochhausen, C., Pfeifer, C. G., Alt, V., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aged+tendon+stem/progenitor+cells+are+less+competent+to+form+3D+tendon+organoids+due+to+cell+autonomous+and+matrix+production+deficits.">Aged tendon stem/progenitor cells are less competent to form 3D tendon organoids due to cell autonomous and matrix production deficits.</a> Front Bioeng Biotechnol. 8, 406 (2020).</li><li>Larkin, L. M., Calve, S., Kostrominova, T. Y., Arruda, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functional+evaluation+of+tendon-skeletal+muscle+constructs+engineered+in+vitro.">Structure and functional evaluation of tendon-skeletal muscle constructs engineered in vitro.</a> Tissue Eng. 12 (11), 3149-3158 (2006).</li><li>Schiele, N. R., Koppes, R. A., Chrisey, D. B., Corr, D. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+uniaxial+tensile+strain+enhances+the+mechanical+properties+of+engineered,+scaffold-free+tendon+fibers.">Cyclic uniaxial tensile strain enhances the mechanical properties of engineered, scaffold-free tendon fibers.</a> Tissue Eng Part A. 24 (23-24), 1808-1817 (2018).</li><li>Brassard, J. A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+stem+cell+self-organization+to+build+better+organoids.">Engineering stem cell self-organization to build better organoids.</a> Cell Stem Cell. 24 (6), 860-876 (2019).</li><li>Kim, J., Koo, B. K., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organoids:+model+systems+for+human+biology+and+medicine.">Human organoids: model systems for human biology and medicine.</a> Nat Rev Mol Cell Biol. 21 (10), 571-584 (2020).</li><li>Hsieh, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+comparison+of+2D-cell+culture+and+3D-cell+sheets+of+scleraxis-programmed+bone+marrow+derived+mesenchymal+stem+cells+to+primary+tendon+stem/progenitor+cells+for+tendon+repair.">In vitro comparison of 2D-cell culture and 3D-cell sheets of scleraxis-programmed bone marrow derived mesenchymal stem cells to primary tendon stem/progenitor cells for tendon repair.</a> Int J Mol Sci. 19 (8), 2272 (2018).</li><li>Chu, J., Lu, M., Pfeifer, C. G., Alt, V., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuilding+tendons:+A+concise+review+on+the+potential+of+dermal+fibroblasts.">Rebuilding tendons: A concise review on the potential of dermal fibroblasts.</a> Cells. 9 (9), 2047 (2020).</li><li>Gaspar, D., Spanoudes, K., Holladay, C., Pandit, A., Zeugolis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+cell-based+therapies+for+tendon+repair.">Progress in cell-based therapies for tendon repair.</a> Adv Drug Deliv Rev. 84, 240-256 (2015).</li><li>Sacco, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+dermal+fibroblasts+as+major+determinant+of+variability+in+cell+reprogramming.">Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming.</a> J Cell Mol Med. 23 (6), 4256-4268 (2019).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+predominant+tenogenic+phenotype+in+human+dermal+fibroblasts+via+synergistic+effect+of+TGF-&#946;+and+elongated+cell+shape.">Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-&#946; and elongated cell shape.</a> Am J Physiol Cell Physiol. 310 (5), C357-C372 (2016).</li><li>Pryce, B. A., Watson, S. S., Murchison, N. D., Staverosky, J. A., D&#252;nker, N., Schweitzer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+and+maintenance+of+tendon+progenitors+by+TGFbeta+signaling+are+essential+for+tendon+formation.">Recruitment and maintenance of tendon progenitors by TGFbeta signaling are essential for tendon formation.</a> Development. 136 (8), 1351-1361 (2009).</li><li>Pattappa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physioxia+has+a+beneficial+effect+on+cartilage+matrix+production+in+interleukin-1+beta-inhibited+mesenchymal+stem+cell+chondrogenesis.">Physioxia has a beneficial effect on cartilage matrix production in interleukin-1 beta-inhibited mesenchymal stem cell chondrogenesis.</a> Cells. 8 (8), 936 (2019).</li><li>Shafiee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+physiologically+relevant+skin+organoids+from+human+induced+pluripotent+stem+cells.">Development of physiologically relevant skin organoids from human induced pluripotent stem cells.</a> Small. 2304879, (2023).</li><li>Zeiger, A. 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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+administration+of+ASCs+and+BMP-12+promotes+an+M2+macrophage+phenotype+and+enhances+tendon+healing.">Combined administration of ASCs and BMP-12 promotes an M2 macrophage phenotype and enhances tendon healing.</a> Clin Orthop Relat Res. 475 (9), 2318-2331 (2017).</li><li>Hofer, M., Lutolf, M. 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Resultatene demonstrert i denne studien gir verdifull innsikt i etablering og karakterisering av NHDF 3D organoidmodell for å studere T / L-vev. 3-trinns protokollen førte til dannelsen av 3D-stavlignende organoider som viser typiske trekk ved T / L-nisje. Denne modellen er tidligere omtalt i Kroner-Weigl et al. 20237 og demonstrert i detalj her.
Fasekontrastbildene presentert i figur 2 viste progresjonen av NHDF-konfluens og ark...

Sener og leddbånd (T/L) er viktige komponenter i muskel- og skjelettsystemet som gir viktig støtte og stabilitet til kroppen. Til tross for deres kritiske rolle, er disse bindevevene utsatt for degenerasjon og skade, noe som forårsaker smerte og nedsatt mobilitet1. Videre kan deres begrensede blodtilførsel og langsomme helbredende kapasitet føre til kroniske skader, mens faktorer som aldring, repeterende bevegelse og feil rehabilitering ytterligere øker risikoen for degenerasjon og skade2. Konvensjonelle behandlinger, som hvile, fysioterapi og kirurgiske inngrep, er ikke i stand til å gjenopprette T / L-strukturen og funksjonen fullt ut. I løpet av de siste årene har forskere forsøkt å bedre forstå den intrikate naturen til T / L for å søke effektive behandlinger for T / L-lidelser 3,4,5. T/L kjennetegnes av en hierarkisk organisert, ekstracellulær matrise (ECM)-dominert struktur, hovedsakelig sammensatt av type I kollagenfibre og proteoglykaner, en egenskap som er vanskelig å replikere in vitro6. Tradisjonelle todimensjonale (2D) cellekulturmodeller klarer ikke å fange den karakteristiske tredimensjonale (3D) organisasjonen av T / L-vev, noe som begrenser deres translasjonspotensial, samt hindrer den innovative fremgangen innen T / L-regenerering.
Nylig har utviklingen av 3D-organoidmodeller gitt nye muligheter for å fremme grunnforskning og stillasfri vevsteknikk av ulike vevstyper 7,8,9,10,11,12,13. For eksempel, for å undersøke myotendinøs kryss, utviklet Larkin et al. 2006 3D-skjelettmuskelkonstruksjoner sammen med selvorganiserte senesegmenter avledet fra rottehalesene10. Videre regisserte Schiele et al. 2013, ved å bruke mikromaskinerte fibronektinbelagte vekstkanaler, selvmontering av humane dermale fibroblaster for å danne cellulære fibre uten hjelp av 3D-stillas, en tilnærming som kan fange viktige trekk ved embryonal seneutvikling11. I studien av Florida et al. 2016 ble benmargsstromale celler først utvidet til bein- og ligamentlinjer, deretter brukt til å generere selvmonterte monolagscelleark, som deretter ble implementert for å lage en multifasisk bein-ligament-beinkonstruksjon som etterligner det innfødte fremre korsbåndet, en modell som tar sikte på forbedret forståelse av ligamentregenerering12. For å belyse senemekanotransduksjonsprosesser benyttet Mubyana et al. 2018 en stillasfri metodikk der enkeltsenefibre ble opprettet og utsatt for mekanisk belastningsprotokoll13. Organoider er selvorganiserte 3D-strukturer som etterligner den opprinnelige arkitekturen, mikromiljøet og funksjonaliteten til vev. 3D organoidkulturer gir en mer fysiologisk relevant modell for å studere vevs- og organbiologi samt patofysiologi. Slike modeller kan også brukes til å indusere vevsspesifikk differensiering av forskjellige stam-/stamcelletyper14,15. Derfor blir implementering av 3D-organoidmodeller innen T / L-biologi og vevsteknikk en veldig attraktiv tilnærming 9,16. Alternative cellekilder kan implementeres for organoidforsamlingen og stimuleres mot tenogen differensiering. En relevant celletype som brukes til demonstrasjon i denne studien er dermale fibroblaster 7,17,18. Disse cellene er lett tilgjengelige gjennom en hudbiopsiprosedyre, som er mindre invasiv sammenlignet med benmargpunktering eller fettsuging og kan multipliseres ganske raskt til store mengder på grunn av deres gode proliferative kapasitet. I motsetning til dette er mer spesialiserte celletyper, som T / L-residente fibroblaster, mer utfordrende å isolere og utvide. Derfor ble dermal fibroblaster også brukt som utgangspunkt for celleomprogrammeringsteknologier mot induserte pluripotente embryonale stamceller19. Å utsette dermale fibroblaster for spesifikke 3D-kulturforhold og signalsignaler, for eksempel transformerende vekstfaktor-beta 3 (TGFß3), som har blitt rapportert å fungere som en nøkkelregulator for forskjellige cellulære prosesser, inkludert dannelse og vedlikehold av T / L, kan potensere deres in vitro tenogene differensiering som fører til ekspresjon av senespesifikke gener og avsetning av T / L-typisk ECM20, 21.
Her beskriver og demonstrerer vi en tidligere etablert og implementert 3-trinns (2D-utvidelse, 2D-stimulering og 3D-modning) organoidprotokoll ved bruk av kommersielt tilgjengelige normale voksne humane dermal fibroblaster (NHDF) som cellekilde, og tilbyr en verdifull modell for å studere in vitro tenogenese7. Til tross for at denne modellen ikke er ekvivalent med in vivo T/L-vev, gir den fortsatt et mer fysiologisk relevant system som kan brukes til å undersøke cellulære differensieringsmekanismer, etterligne T/L-patofysiologi in vitro og etablere T/L-persontilpasset medisin og legemiddelscreeningsplattformer. Videre kan studier i fremtiden evaluere om 3D-organoidene er egnet for stillasfri T / L-prosjektering ved ytterligere optimalisering, samt brukbar for utvikling av oppskalerte mekanisk robuste konstruksjoner som ligner dimensjonene og strukturelle og biofysiske egenskapene til innfødte T / L-vev.

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			<td>Ascorbic acid&#160;&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;&#160;</td>
			<td>A8960</td>
			<td></td>
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		<tr>
			<td>10 cm adherent cell culture dish</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430167</td>
			<td></td>
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		<tr>
			<td>10 cm non-adherent petri dish&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430591</td>
			<td></td>
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			<td>Cryo-medium</td>
			<td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands&#160;&#160;</td>
			<td>4583</td>
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			<td>Cryomold standard&#160;</td>
			<td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands</td>
			<td>4557</td>
			<td></td>
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			<td>D(+)-Sucrose&#160;</td>
			<td>AppliChem Avantor VWR International GmbH, Darmstadt, Germany</td>
			<td>A2211</td>
			<td></td>
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			<td>DMEM high glucose medium&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>DMEM-HA</td>
			<td></td>
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			<td>DMEM low glucose</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>DMEM-LPXA</td>
			<td></td>
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			<td>Fetal bovine serum</td>
			<td>&#160;Anprotec, Bruckberg, Germany&#160;</td>
			<td>AC-SM-0027</td>
			<td></td>
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			<td>Fibroblast growth medium 2&#160;</td>
			<td>PromoCell, Heidelberg, Germany&#160;&#160;</td>
			<td>C-23020</td>
			<td></td>
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			<td>Inverted microscope with high resolution camera</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td>Zeiss Axio Observer with&#160; Axiocam 506</td>
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			<td>MEM amino acids&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>NEAA-B</td>
			<td></td>
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			<td>Metal pins&#160;</td>
			<td>EntoSphinx, Pardubice, Czech Republic&#160;</td>
			<td>04.31</td>
			<td></td>
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			<td>Normal human dermal fibroblasts&#160;</td>
			<td>&#160;PromoCell, Heidelberg, Germany&#160;</td>
			<td>C-12302</td>
			<td></td>
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			<td>Paraformaldehyde</td>
			<td>&#160;AppliChem, Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>A3813</td>
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			<td>Penicillin/streptomycin&#160;</td>
			<td>Gibco, Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>15140122</td>
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			<td>Phosphate buffer saline&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>P4417</td>
			<td></td>
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			<td>TGF&#223;3&#160;</td>
			<td>R&#38;D Systems, Wiesbaden, Germany&#160;</td>
			<td>&#160;8420-B3</td>
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			<td>Trypsin-EDTA 0,05% DPBS&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>&#160;TRY-1B</td>
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demonstration of self-assembled cell sheet culture and manual generation of a 3d tendon/ligament-like organoid by using human dermal fibroblasts

Sener og leddbånd (T/L) er sterke hierarkisk organiserte strukturer som forener muskel- og skjelettsystemet. Disse vevene har en strengt arrangert kollagen type I-rik ekstracellulær matrise (ECM) og T / L-avstamningsceller hovedsakelig plassert i parallelle rader. Etter skade krever T/L lang tid for rehabilitering med høy sviktrisiko og ofte utilfredsstillende reparasjonsresultater. Til tross for nylige fremskritt innen T / L-biologisk forskning, er en av de gjenværende utfordringene at T / L-feltet fortsatt mangler en standardisert differensieringsprotokoll som er i stand til å rekapitulere T / L-dannelsesprosessen in vitro. For eksempel krever ben- og fettdifferensiering av mesenkymale forløperceller bare standard todimensjonal (2D) cellekultur og tilsetning av spesifikke stimuleringsmedier. For differensiering til brusk er tredimensjonal (3D) pelletskultur og tilskudd av TGFß nødvendig. Imidlertid trenger celledifferensiering til sene en veldig ryddig 3D-kulturmodell, som ideelt sett også bør utsettes for dynamisk mekanisk stimulering. Vi har etablert en 3-trinns (ekspansjon, stimulering og modning) organoidmodell for å danne en 3D-stavlignende struktur ut av et selvmontert celleark, som leverer et naturlig mikromiljø med sine egne ECM-, autokrine og parakrine faktorer. Disse stavlignende organoidene har en flerlags cellulær arkitektur i rik ECM og kan håndteres ganske enkelt for eksponering for statisk mekanisk belastning. Her demonstrerte vi 3-trinns protokollen ved å bruke kommersielt tilgjengelige dermale fibroblaster. Vi kunne vise at denne celletypen danner robuste og ECM-rike organoider. Den beskrevne prosedyren kan optimaliseres ytterligere når det gjelder kulturmedier og optimaliseres mot dynamisk aksial mekanisk stimulering. På samme måte kan alternative cellekilder testes for deres potensial til å danne T / L-organoider og dermed gjennomgå T / L-differensiering. I sum kan den etablerte 3D T / L organoide tilnærmingen brukes som en modell for senegrunnforskning og til og med for stillasfri T / L-engineering.

Tendons and ligaments (T/L) are vital components of the musculoskeletal system that provide essential support and stability to the body. Despite their critical role, these connective tissues are prone to degeneration and injury, causing pain and impairment of mobility1. Moreover, their limited blood supply and slow healing capacity can lead to chronic injuries, whereas factors such as aging, repetitive motion, and improper rehabilitation further increase the risk of degeneration and injury2. Conventional treatments, such as rest, physical therapy, and surgical interventions, are unable to fully restore the T/L structure and function. Over the past few years, researchers have strived to better understand the intricate nature of T/L in order to seek effective treatments for T/L disorders3,4,5. T/L are distinguished by a hierarchically organized, extracellular matrix (ECM)-dominated structure, composed primarily of type I collagen fibers and proteoglycans, a feature that is difficult to be replicated in vitro6. Traditional two-dimensional (2D) cell culture models fail to capture the characteristic three-dimensional (3D) organization of T/L tissues, limiting their translational potential as well as hindering the innovative progress in the field of T/L regeneration.
Recently, the development of 3D organoid models has offered new possibilities for advancing basic research and scaffolds-free tissue engineering of various tissue types7,8,9,10,11,12,13. For example, to investigate myotendinous junction, Larkin et al. 2006 developed 3D skeletal muscle constructs together with self-organized tendon segments derived from rat tail tendon10. Moreover, Schiele et al. 2013, by using micromachined fibronectin-coated growth channels, directed the self-assembly of human dermal fibroblasts to form cellular fibers without the assistance of 3D scaffold, an approach that can capture key traits of embryonic tendon development11. In the study by Florida et al. 2016, bone marrow stromal cells were first expanded into bone and ligament lineages, next used to generate self-assembled monolayer cell sheets, which were then implemented to create a multiphasic bone-ligament-bone construct mimicking the native anterior cruciate ligament, a model aiming at improved understanding of ligament regeneration12. To elucidate tendon mechanotransduction processes, Mubyana et al. 2018 utilized a scaffold-free methodology by which single tendon fibers were created and subjected to mechanical loading protocol13. Organoids are self-organized 3D structures that mimic the native architecture, microenvironment, and functionality of tissues. 3D organoid cultures provide a more physiologically relevant model for studying tissue and organ biology as well as pathophysiology. Such models can also be used to induce tissue-specific differentiation of different stem/progenitor cell types14,15. Hence, implementing 3D organoid models in the field of T/L biology and tissue engineering becomes a very attractive approach9,16. Alternative cell sources can be implemented for the organoid assembly and stimulated toward tenogenic differentiation. One relevant cell type used for demonstration in this study is dermal fibroblasts7,17,18. These cells are easily accessible through a skin biopsy procedure, which is less invasive compared to bone marrow puncture or liposuction and can be multiplied fairly quickly to large numbers due to their good proliferative capacity. In contrast, more specialized cell types, such as T/L-resident fibroblasts, are more challenging to isolate and expand. Therefore, dermal fibroblasts were also used as a starting point for cell reprogramming technologies towards induced pluripotent embryonic stem cells19. Subjecting dermal fibroblasts to specific 3D culture conditions and signaling cues, such as transforming growth factor-beta 3 (TGF&#223;3), which has been reported to act as a key regulator of various cellular processes, including the formation and maintenance of T/L, can potentiate their in vitro tenogenic differentiation leading to the expression of tendon-specific genes and the deposition of T/L-typical ECM20,21.
Here, we describe and demonstrate a previously established and implemented 3-step (2D expansion, 2D stimulation, and 3D maturation) organoid protocol using commercially available normal adult human dermal fibroblasts (NHDFs) as a cell source, offering a valuable model for studying in vitro tenogenesis7. Despite the fact that this model is not equivalent to in vivo T/L tissue, it still provides a more physiologically relevant system that can be used for investigating cellular differentiation mechanisms, mimicking T/L pathophysiology in vitro, and establishing T/L personalized medicine and drug screening platforms. Moreover, in the future, studies can evaluate whether the 3D organoids are suitable for scaffold-free T/L engineering by further optimization as well as utilizable for the development of scaled-up mechanically robust constructs that closely resemble the dimensions and structural and biophysical properties of native T/L tissues.

Forfatter i søkelyset: Fremme senevevsteknikk med 3D-organoidmodeller

Demonstrasjon av selvmontert cellearkkultur og manuell generering av en 3D-sene / ligamentlignende organoid ved bruk av humane dermale fibroblaster

We demonstrate a previous established and implement three-step organoid protocol using commercially available, normal human dermal fibroblasts as a cell source. The 3D organoid model holds several advantage for tissue engineering and regenerative medicine application. It mimics closely the cellular composition and organization as well as cell-to-cell and cell-to-matrix interactions of tendon tissues.The established 3D tendon-ligament organoid approach can be used as a model for tendon basic and applied research in the following areas. In vitro tenogenesis, disease mechanism, drug testing, and scaffold-free tendon tissue engineering.

3D T/L organoidmodellen ble tidligere etablert og demonstrert her ved å implementere kommersielt innkjøpt NHDF (n=3, 3 organoider per donor, NHDF ble brukt i passasje 5-8). Modellarbeidsflyten er oppsummert i figur 1. Figur 2 viser representative fasekontrastbilder av NHDF-kultur under pre-ekspansjonen i T-75-kolber (figur 2A) samt i begynnelsen og etter 5 dager med kultur i 2D-ekspansjonstrinnet i 10 cm cellekulturskåler (<stron...

Her demonstrerer vi en tre-trinns organoidmodell (todimensjonal [2D] ekspansjon, 2D-stimulering, tredimensjonal [3D] modning) som tilbyr et lovende verktøy for senegrunnforskning og en potensiell stillasfri metode for senevevsteknikk.

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

Vi demonstrerer en tidligere etablert og implementerer tre-trinns organoidprotokoll ved bruk av kommersielt tilgjengelige, normale humane dermale fibroblaster som cellekilde. 3D-organoidmodellen har flere fordeler for vevsteknikk og regenerativ medisinapplikasjon. Det etterligner nøye cellulær sammensetning og organisasjon, samt celle-til-celle og celle-til-matrise-interaksjoner av senevev.Den etablerte 3D sene-ligament organoid tilnærming kan brukes som en modell for sene grunnleggende og anvendt forskning på følgende områder. In vitro tenogenese, sykdomsmekanisme, medikamenttesting og stillasfri senevevsteknikk.

demonstration-self-assembled-cell-sheet-culture-manual-generation-3d

Research

JoVE Journal

Bioengineering

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly arranged collagen type&#160;I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGF&#223; is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Herein, we demonstrate a three-step organoid model (two-dimensional [2D] expansion, 2D stimulation, three-dimensional [3D] maturation) offering a promising tool for tendon fundamental research and a potential scaffold-free method for tendon tissue engineering.