Name: Forfatter Spotlight: Fremme af senevævsteknik 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. anerkende BMBF-bevillingen "CellWiTaL: Reproducerbare cellesystemer til lægemiddelforskning - overførselslagfri laserudskrivning af meget specifikke enkeltceller i tredimensionelle cellulære strukturer" Forslag nr. 13N15874. D.D. og V.R.A. anerkender EU MSCA-COFUND Grant OSTASKILLS "Holistisk træning af næste generations slidgigtforskning" GA Nr. 101034412. Alle forfattere anerkender fru Beate Geyer for teknisk assistance.

BEMÆRK: Alle trin skal udføres ved hjælp af aseptiske teknikker.
1. Kultur og præ-udvidelse af NHDF'er
<ol><li>Kryohætteglasset, der indeholder voksne kryopræserverede normale humane dermale fibroblaster (NHDF'er, 1 x 106 celler) ved 37 °C, tøs hurtigt op, indtil de næsten optøes.</li>
	<li>Tilsæt langsomt 1 ml forvarmet fibroblastvækstmedium 2 (brugsklart sæt inklusive basalmedium, 2% føtalt kalveserum (FCS), basisk fibroblastvækstfaktor (bFGF) og ...

Tre-trins organoidmodel til seneforskning og -teknik

<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. 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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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Resultaterne demonstreret i denne undersøgelse giver værdifuld indsigt i etablering og karakterisering af NHDF 3D organoid model til undersøgelse af T / L væv. 3-trinsprotokollen førte til dannelsen af 3D-stanglignende organoider, der udviser typiske træk ved T / L-niche. Denne model er tidligere omtalt i Kroner-Weigl et al. 20237 og demonstreret meget detaljeret her.
Fasekontrastbillederne vist i figur 2 viste progressionen ...

Sener og ledbånd (T / L) er vitale komponenter i muskuloskeletalsystemet, der giver væsentlig støtte og stabilitet til kroppen. På trods af deres kritiske rolle er disse bindevæv tilbøjelige til degeneration og skade, hvilket forårsager smerte og svækkelse af mobilitet1. Desuden kan deres begrænsede blodforsyning og langsomme helingskapacitet føre til kroniske skader, mens faktorer som aldring, gentagne bevægelser og forkert rehabilitering yderligere øger risikoen for degeneration og skade2. Konventionelle behandlinger, såsom hvile, fysioterapi og kirurgiske indgreb, er ikke i stand til fuldt ud at genoprette T / L-strukturen og funktionen. I løbet af de sidste par år har forskere stræbt efter bedre at forstå den indviklede karakter af T / L for at søge effektive behandlinger for T / L lidelser 3,4,5. T / L er kendetegnet ved en hierarkisk organiseret, ekstracellulær matrix (ECM) -domineret struktur, der primært består af type I kollagenfibre og proteoglycaner, en funktion, der er vanskelig at replikere in vitro6. Traditionelle todimensionelle (2D) cellekulturmodeller fanger ikke den karakteristiske tredimensionelle (3D) organisering af T / L-væv, hvilket begrænser deres translationelle potentiale samt hindrer de innovative fremskridt inden for T / L-regenerering.
For nylig har udviklingen af 3D-organoidmodeller givet nye muligheder for at fremme grundforskning og stilladsfri vævsteknik af forskellige vævstyper 7,8,9,10,11,12,13. For eksempel for at undersøge myotendinøs krydsning udviklede Larkin et al. 2006 3D-skeletmuskelkonstruktioner sammen med selvorganiserede senesegmenter afledt af rottehalesenen10. Desuden dirigerede Schiele et al. 2013 ved hjælp af mikrobearbejdede fibronectinbelagte vækstkanaler selvmontering af humane dermale fibroblaster til dannelse af cellulære fibre uden hjælp fra 3D-stillads, en tilgang, der kan fange nøgletræk ved embryonal seneudvikling11. I undersøgelsen af Florida et al. 2016 blev knoglemarvstromale celler først udvidet til knogle- og ligamentlinjer, der derefter blev brugt til at generere selvmonterede enkeltlagscelleark, som derefter blev implementeret for at skabe en multifasisk knogle-ligament-knoglekonstruktion, der efterligner det indfødte forreste korsbånd, en model, der sigter mod forbedret forståelse af ligamentregenerering12. For at belyse senemekanotransduktionsprocesser anvendte Mubyana et al. 2018 en stilladsfri metode, hvormed enkelte senefibre blev skabt og udsat for mekanisk belastningsprotokol13. Organoider er selvorganiserede 3D-strukturer, der efterligner vævets oprindelige arkitektur, mikromiljø og funktionalitet. 3D organoidkulturer giver en mere fysiologisk relevant model til at studere vævs- og organbiologi samt patofysiologi. Sådanne modeller kan også anvendes til at inducere vævsspecifik differentiering af forskellige stam-/stamcelletyper14,15. Derfor bliver implementering af 3D-organoidmodeller inden for T / L-biologi og vævsteknik en meget attraktiv tilgang 9,16. Alternative cellekilder kan implementeres til organoidsamlingen og stimuleres mod tenogen differentiering. En relevant celletype, der anvendes til demonstration i denne undersøgelse, er dermale fibroblaster 7,17,18. Disse celler er let tilgængelige gennem en hudbiopsiprocedure, som er mindre invasiv sammenlignet med knoglemarvspunktur eller fedtsugning og kan multipliceres ret hurtigt til store tal på grund af deres gode proliferative kapacitet. I modsætning hertil er mere specialiserede celletyper, såsom T / L-residente fibroblaster, mere udfordrende at isolere og udvide. Derfor blev dermale fibroblaster også anvendt som udgangspunkt for celleprogrammeringsteknologier til inducerede pluripotente embryonale stamceller19. At udsætte dermale fibroblaster for specifikke 3D-kulturbetingelser og signalsignaler, såsom transformering af vækstfaktor-beta 3 (TGFß3), som er rapporteret at fungere som en nøgleregulator for forskellige cellulære processer, herunder dannelse og vedligeholdelse af T/L, kan forstærke deres in vitro tenogene differentiering, hvilket fører til ekspression af senespecifikke gener og deponering af T/L-typisk ECM20, 21.
Her beskriver og demonstrerer vi en tidligere etableret og implementeret 3-trins (2D-ekspansion, 2D-stimulering og 3D-modning) organoidprotokol ved hjælp af kommercielt tilgængelige normale voksne humane dermale fibroblaster (NHDF'er) som cellekilde, der tilbyder en værdifuld model til undersøgelse af in vitro tenogenese7. På trods af at denne model ikke svarer til in vivo T / L-væv, giver den stadig et mere fysiologisk relevant system, der kan bruges til at undersøge cellulære differentieringsmekanismer, efterligne T / L patofysiologi in vitro og etablere T / L personlig medicin og lægemiddelscreeningsplatforme. Desuden kan undersøgelser i fremtiden evaluere, om 3D-organoiderne er egnede til stilladsfri T/L-konstruktion ved yderligere optimering samt anvendelige til udvikling af opskalerede mekanisk robuste konstruktioner, der ligner dimensionerne og strukturelle og biofysiske egenskaber af naturligt T/L-væv.

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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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			<td>10 cm adherent cell culture dish</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430167</td>
			<td></td>
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			<td>10 cm non-adherent petri dish&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430591</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>
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			<td>D(+)-Sucrose&#160;</td>
			<td>AppliChem Avantor VWR International GmbH, Darmstadt, Germany</td>
			<td>A2211</td>
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			<td>DMEM high glucose medium&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>DMEM-HA</td>
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			<td>DMEM low glucose</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>DMEM-LPXA</td>
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			<td>Fetal bovine serum</td>
			<td>&#160;Anprotec, Bruckberg, Germany&#160;</td>
			<td>AC-SM-0027</td>
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			<td>Fibroblast growth medium 2&#160;</td>
			<td>PromoCell, Heidelberg, Germany&#160;&#160;</td>
			<td>C-23020</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>
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			<td>Metal pins&#160;</td>
			<td>EntoSphinx, Pardubice, Czech Republic&#160;</td>
			<td>04.31</td>
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			<td>Normal human dermal fibroblasts&#160;</td>
			<td>&#160;PromoCell, Heidelberg, Germany&#160;</td>
			<td>C-12302</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>Phosphate buffer saline&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>P4417</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 ledbånd (T / L) er stærke hierarkisk organiserede strukturer, der forener muskuloskeletalsystemet. Disse væv har en strengt arrangeret kollagen type I-rig ekstracellulær matrix (ECM) og T / L-afstamningsceller hovedsageligt placeret i parallelle rækker. Efter skade kræver T / L lang tid til rehabilitering med høj fejlrisiko og ofte utilfredsstillende reparationsresultater. På trods af de seneste fremskridt inden for T / L biologiforskning er en af de resterende udfordringer, at T / L-feltet stadig mangler en standardiseret differentieringsprotokol, der er i stand til at rekapitulere T / L-dannelsesprocessen in vitro. For eksempel kræver knogle- og fedtdifferentiering af mesenkymale forløberceller kun standard todimensionel (2D) cellekultur og tilsætning af specifikke stimuleringsmedier. For differentiering til brusk er tredimensionel (3D) pelletkultur og tilskud af TGFß nødvendig. Imidlertid kræver celledifferentiering til sener en meget ordnet 3D-kulturmodel, som ideelt set også bør kunne udsættes for dynamisk mekanisk stimulering. Vi har etableret en 3-trins (ekspansion, stimulering og modning) organoidmodel til dannelse af en 3D-stanglignende struktur ud af et selvmonteret celleark, som leverer et naturligt mikromiljø med sine egne ECM-, autokrine- og parakrinefaktorer. Disse stanglignende organoider har en flerlags cellulær arkitektur inden for rig ECM og kan håndteres ganske let til eksponering for statisk mekanisk belastning. Her demonstrerede vi 3-trinsprotokollen ved hjælp af kommercielt tilgængelige dermale fibroblaster. Vi kunne vise, at denne celletype danner robuste og ECM-rigelige organoider. Den beskrevne procedure kan optimeres yderligere med hensyn til kulturmedier og optimeres mod dynamisk aksial mekanisk stimulering. På samme måde kan alternative cellekilder testes for deres potentiale til at danne T / L-organoider og dermed gennemgå T / L-differentiering. Alt i alt kan den etablerede 3D T / L organoid tilgang bruges som en model for senegrundforskning og endda til stilladsfri T / L-teknik.

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 Spotlight: Fremme af senevævsteknik med 3D-organoidmodeller

Demonstration af selvsamlet cellearkkultur og manuel generering af en 3D-sene-/ledbåndslignende organoid ved hjælp af 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 blev tidligere etableret og demonstreret her ved implementering af kommercielt indkøbt NHDF (n=3, 3 organoider pr. donor, NHDF blev brugt ved passager 5-8). Modelarbejdsgangen er opsummeret i figur 1. Figur 2 viser repræsentative fasekontrastbilleder af NHDF-kultur under præekspansion i T-75-kolber (figur 2A) samt i begyndelsen og efter 5 dages dyrkning i 2D-ekspansionstrinnet i 10 cm cellekulturskåle (<s...

Heri demonstrerer vi en tre-trins organoidmodel (todimensionel [2D] ekspansion, 2D-stimulering, tredimensionel [3D] modning), der tilbyder et lovende værktøj til senegrundforskning og en potentiel stilladsfri metode til senevævsteknik.

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 etableret og implementerer tre-trins organoidprotokol ved hjælp af kommercielt tilgængelige, normale humane dermale fibroblaster som cellekilde. 3D-organoidmodellen har flere fordele til vævsteknik og regenerativ medicinanvendelse. Det efterligner nøje den cellulære sammensætning og organisation samt celle-til-celle og celle-til-matrix interaktioner af senevæv.Den etablerede 3D sene-ligament organoid tilgang kan bruges som model for senegrundforskning og anvendt forskning inden for følgende områder. In vitro tenogenese, sygdomsmekanisme, lægemiddeltest og stilladsfri senevævsteknik.

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.