Questo progetto è stato finanziato dal NIH (R21EB013717) e IUPUI OVCR (RSFG). L&#39;autore ringrazia la signora Han Shih per la sua assistenza tecnica.

A. macromero e sintesi peptidica <ol><li> Sintetizzare 4 bracci PEG-norbornene (PEG4NB) e litio arylphosphanate fotoiniziatore (LAP) utilizzando protocolli stabiliti. 18,19 </li><li> Sintetizzare bis-cisteina contenente chimotripsina sensibile peptide C CGGY ↓ (freccia indica sito di taglio della chimotripsina) con standard di sintesi peptidica in fase solida in un sintetizzatore di peptidi a microonde (CEM Discover SPS). <ol class="lalpha"><li> Calcolare la quantità di resina (resina Rink amide-MBHA) richiesta in base al rapporto di sostituzione della resina e la scala di sintesi. Rigonfiare la resina in dimetilformammide (DMF) in un recipiente di reazione (RV) per 15 min. </li><li> Rimuovere Fmoc-gruppo protettivo dalla resina con una soluzione di deprotezione contenente 20% Piperidina e 0,1 M HOBt in DMF. Utilizzare i parametri elencati nella tabella qui sotto per micro-onde Fmoc-deprotezione (per tutti i Fmoc-amminoacidi): <table border="1"><tbody><tr><td> Sintesi scala </strong&gt; </td><td> 0,1 mmoli </td><td> 0,2 mmoli </td></tr><tr><td> Potenza </td><td> 20 W </td><td> 50 W </td></tr><tr><td> Temperatura </td><td> 75 ° C </td><td> 75 ° C </td></tr><tr><td> Tempo </td><td> 3 min </td><td> 3 min </td></tr></tbody></table></li><li> Seguendo deprotezione, lavare la resina con DMF ed eseguire test della ninidrina per confermare la rimozione del Fmoc (esposizione di ammina N-terminale). Resina dovrebbe diventare blu dopo 2 minuti a 95 ° C. </li><li> Dopo deprotezione con successo, la prima coppia Fmoc-amminoacido al C-terminale (sintesi va da C a N-terminale) in una soluzione di base attivatore (0,28 M Diisopropiletilammina (DIEA) in DMF) con i parametri elencati nella tabella seguente . <table border="1"><tbody><tr><td> Sintesi scala </td><td> 0,1 mmoli </td><td> 0,2 mmoli </td&gt; </tr><tr><td> Potenza </td><td> 20 W </td><td> 50 W </td></tr><tr><td> Temperatura * </td><td> 75 ° C </td><td> 75 ° C </td></tr><tr><td> Ora * </td><td> 5 min </td><td> 5 min </td></tr></tbody></table> * Per cisti e la sua, utilizzare 50 ° C e 10 min per ridurre racemizzazione. </li><li> Risciacquare la resina ed eseguire test della ninidrina per confermare il completamento della qualitativamente accoppiamento (scomparsa di ammina N-terminale). Resina dovrebbe essere chiaro dopo 5 min a 95 ° C. Ripetere il punto di accoppiamento (A.2.d) se il test restituisce ninidrina risultato positivo (colore blu). </li><li> Ripetere deprotezione (passo A.2.b) e giunto (A.2.d passo) per ulteriori amminoacidi nella sequenza che termina con un passo deprotezione finale. </li><li> Scolare la RV, sciacquare resina con 25 ml di diclorometano (DCM). Questo passo è per sciacquare DMF, che è una base e ostacolerà clivaggio del peptide. </li><li> Preparare cocktail scissione mescolando 250 mfenolo in 4,75 ml di acido trifluoroacetico (TFA), 0,125 ml triisopropilsilano (TIS) e 0,125 ml DDH 2 O. g </li><li> Aggiungere la soluzione di scissione nel camper e si unirà per 30 minuti nel forno a microonde sintetizzatore di peptidi (Potenza = 20 W, temperatura = 38 ° C). </li><li> Raccogliere la soluzione del peptide in un tubo da centrifuga da 50 ml con collettore di vuoto. </li><li> Peptide precipitato spaccati in 40 ml di etere, vortex la provetta e centrifugare a 3000 rpm per 5 minuti per raccogliere il peptide. </li><li> Scolare il surnatante e ripetere A.2.k passaggio due volte. </li><li> Raccogliere e asciugare peptide nel vuoto. </li><li> Purificare il peptide mediante HPLC e caratterizzare con la spettrometria di massa. </li></ol></li></ol> B. Preparazione del materiale e sterilizzazione <ol><li> Preparare 20 wt soluzione PEG4NB% in PBS, vortex la miscela finché il macromero dissolve completamente, e sterilizzare la soluzione macromero utilizzando un filtro siringa. Conservare la soluzione sterilizzata a -20 ° C (preparare aliquoteper la conservazione a lungo termine). </li><li> Preparare e sterilizzare due fotoiniziatore% in peso (LAP) soluzione in PBS. Conservare la soluzione sterilizzata a temperatura ambiente al riparo dalla luce. </li><li> Sciogliere il peptide (CGGYC) in PBS, vortex la miscela e siringa filtrare la soluzione per la sterilizzazione. Dispensare la soluzione di peptide e conservare a -20 ° C fino all&#39;uso (evitare cicli gelo-disgelo). </li><li> Determinare tiolo (-SH) concentrazione della soluzione preparata peptide usando il reagente di Ellman (seguire il protocollo del produttore). Questo passo darà accurata concentrazione di peptide (cioè, [SH] / 2) che è richiesto per calcolare l&#39;importo peptide da utilizzare in tiolo-ene fotografica clic reazioni. </li><li> Preparare stampi gelificazione tagliando la parte superiore fuori di sterili monouso siringhe da 1 ml con una lama di rasoio (punta aperta per la rimozione di gel). Sterilizzare gli stampi siringa in autoclave. </li><li> Basato sulla formulazione gel desiderato (tiolo rapporto ene = 1) e le concentrazioni di mate magazzinoriali, calcolare il volume richiesto di polimero, reticolante peptide, fotoiniziatore, e soluzioni tampone richieste. </li></ol> C. Preparazione cellulare <ol><li> Equilibrare terreno di coltura cellulare (alta glucosio DMEM contenente 10% di siero fetale bovino, antibiotici antimicotica con 100 U / ml di penicillina, 100 ug / ml di streptomicina, 250 ng / ml Fungizone, e 50 mM β-mercaptoetanolo), Hank soluzione equilibrata salina ( HBSS, Ca 2 +, Mg 2 + libero), e Tripsina-EDTA a 37 ° C. </li><li> Rimuovere flaconi contenenti MIN6 β-cellule di CO 2 incubatore e porre i matracci in cappa a flusso laminare. </li><li> Aspirare mezzo di coltura dalle beute e sciacquare la cella con HBSS. </li><li> Tripsinizzare le cellule utilizzando 3 ml di 2X (0,1%) Tripsina-EDTA e incubare i flaconi per 4 min a 37 ° C e 5% di CO 2. <ul><li> Nota: Usare una soluzione di tripsina 2X permette di completa dissociazione MIN6 β-cellule in sospensione singola cella. </li></ul> &lt;/ Li&gt; <li> Dopo l&#39;incubazione, toccare le beute sulla superficie della cappa delicatamente per staccare completamente le cellule. Confermare il distacco e la dissociazione delle cellule al microscopio. </li><li> Aggiungere uguale volume di mezzo di coltura cellulare per neutralizzare la tripsina. Mescolare bene la soluzione pipettando dolce, quindi trasferire il composto in una provetta canonica e centrifugare per 5 min a 1.000 giri al minuto. </li><li> Aspirare il surnatante e risospendere delicatamente le cellule in 4-5 ml di terreno di coltura. </li><li> Determinare la densità cellulare utilizzando 50% Trypan blue tampone in un emocitometro. </li></ol> D. Fabbricazione Idrogel e Encapsulation cellulare <ol><li> Una volta che le cellule sono pronte, mix (sulla base di un tiolo rapporto 1:1 ene) i quantitativi necessari di PEG4NB, CGGYC, LAP, soluzione cellulare, e HBSS in una provetta da microcentrifuga. </li><li> Mescolare lentamente la soluzione con una pipetta ad ottenere un impasto omogeneo e aggiungere 25 microlitri della soluzione risultante per uno stampo siringa. Esporre la siringa alla luce UV (365nm, 5 mW / cm 2) per 2 min. </li><li> Dopo la fotopolimerizzazione, gel immergersi in una sterile 24 e mezzo di cella piastra contenente cultura e incubare le idrogel a 37 ° C e 5% di CO 2. </li><li> Lavare le cellule cariche di idrogeli in mezzo di coltura cellulare per 30 minuti per lavare le cellule debolmente collegate e poco reticolati macromeri. Trasferire i gel in terreno fresco. </li><li> Aggiornamento mezzo di coltura ogni 2 o 3 giorni. </li></ol> E. Cella test di vitalità Morfologia cellulare incapsulato può essere osservato mediante microscopio ottico (poiché gli idrogel sintetizzati sono trasparenti). La vitalità cellulare possono essere visualizzati e misurati qualitativa, utilizzando Live / Dead colorazione e quantitativamente usando il reagente AlamarBlue. E.1. Live / Dead colorazione <ol><li> Scongelare Live / Dead reagenti di colorazione a temperatura ambiente. </li><li> In una provetta aggiungere canonica PBS secondo il numero di campioni (n) di essere tinto: <strong&gt; Volume totale di PBS = n × 500 microlitri </li><li> Pipettare 0,25 ml di calceina AM e 2 microlitri di ETHD-1 per 1 ml di PBS al tubo canonica contenente PBS. Vortex la soluzione per miscelare i componenti. </li><li> Incubare cellule cariche di idrogeli in soluzione colorazione Live / Dead (0,5 ml / campione) per 1 ora a temperatura ambiente su un agitatore orbitale. </li><li> Usando una pipetta Pasteur, rimuovere la soluzione di colorante e lavare il campione due volte con PBS. </li><li> Mettere campione su un vetrino coprioggetto o vetrino. Osservare e celle di immagine utilizzando un microscopio confocale. </li></ol> E.2. AlamarBlue Assay <ol><li> Preparare 10 v / v soluzione% AlamarBlue nel mezzo di coltura cellulare. </li><li> Rimuovere idrogel da incubatore e aspirare i supporti su ciascun pozzetto senza alcun contatto con l&#39;idrogel. </li><li> Incubare cellule cariche di idrogel e un vuoto (controllo negativo) e in 500 ml di soluzione al 10% AlamarBlue per 16 ore. </li><li> Dopo l&#39;incubazione, pipettare 200 uldella soluzione AlamarBlue in una piastra a 96 pozzetti e fluorescenza misura utilizzando un lettore per micropiastre (eccitazione: 560 nm, emissione: 590 nm). <ul><li> L&#39;attività metabolica cellulare superiore riduce il colorante per dare maggiore lettura della fluorescenza. </li><li> Assicurati di includere almeno due pozzetti della soluzione al 10% Alamarblue come bianco. Nell&#39;analizzare i dati, sottrarre il valore di bianco fluorescenza mediato dalla lettura della fluorescenza dei campioni. </li></ul></li></ol> F. Chymotrypsin Mediata Erosione Gel e ripristino Spheroid <ol><li> Preparare 1 mg / ml di tripsina-α-chimotripsina libera in soluzione sterile HBBS e filtrare la soluzione. </li><li> Incubare idrogel in soluzione chimotripsina (500 microlitri per ogni gel) a temperatura ambiente agitando delicatamente fino erosione gel viene raggiunto. <ul><li> Per un gel 25 pl, il tempo per la completa erosione è di circa 5 min. </li></ul></li><li> Posizionare la piastra su ghiaccio per alcuni minuti per ridurre l&#39;attività dichimotripsina. </li><li> Trasferire la soluzione cellulare in 1 ml provetta e centrifugare a 300 rpm, 4 ° C per 2 min. </li><li> Rimuovere con attenzione il surnatante con una pipetta pascolo e delicatamente risospendere le sferoidi in HBSS. </li><li> Trasferire la soluzione recuperata sferoide a un 24-pozzetti e posizionare la piastra su ghiaccio per consentire agli sferoidi di stabilirsi. </li><li> Per l&#39;analisi dimensioni, acquisire immagini contrasto di fase delle sferoidi corrisposto tramite un microscopio ottico. Misura recuperato diametri sferoidali che utilizzano software come elemento software Nikon o ImageJ. </li></ol> G. Analisi funzionale di sferoidi recuperati G.1. Glucosio secrezione di insulina stimolata da sferoidi di cellule recuperate <ol><li> In una piastra a 24 pozzetti, incubare i gel in 500 ml di 2 mM glucosio basso Cordoli-Ringer buffer (KRB) per 1 ora. </li><li> Erode il gel e recuperare sferoidi cellulari che utilizzano passi F.1 a F.6. </li><li> Aspirare il surnatante con attenzione senza fare alcunil contatto con gli sferoidi e risospendere le cellule in 500 microlitri sferoidi di 2 KRB mM glucosio basso. </li><li> Trasferire 100 ul di soluzione di sferoidi cellule in una provetta da 0,6 ml e posizionarlo sul ghiaccio per quantificazione dell&#39;ATP intracellulare. </li><li> Dividere la soluzione rimanente cellula sferoidale a metà (in due provette da microcentrifuga) e centrifugare per 2 min a 300 giri al minuto e 4 ° C. </li><li> Aspirare il surnatante (quasi fino al fondo) e risospendere prima metà di sferoidi di cellule in 1 ml di 25 mM di glucosio KRB alta e metà in 1 ml di 2 mM KRB glucosio basso. </li><li> Delicatamente pipetta e trasferire la soluzione contenente sferoidi di cellule recuperate ad un 24-pozzetti. Incubare la piastra per 1 ora a 37 ° C e 5% di CO 2. </li><li> Dopo l&#39;incubazione, trasferire la soluzione in una provetta e centrifugare per 2 min a 1000 rpm e 4 ° C. </li><li> Trasferire 500 pl del supernatante dal tubo centrifugato ad un&#39;altra provetta da microcentrifuga e conservare a 4 ° C per l&#39;insulinaELISA. </li><li> Eseguire protocollo insulina produttore ELISA successivo. </li></ol> G.2. CellTiter Glo Assay <ol><li> Norme ATP: Preparare una soluzione di 10 mM ATP e di eseguire diluizioni seriali per ottenere una serie di 8 soluzioni standard. </li><li> Per un bianco piastra a 96 pozzetti, aggiungere 50 ml di soluzione di cellule sferoide (dal punto G.1.6) e gli standard. </li><li> Per ciascun campione pozzetto contenente e standard, aggiungere 50 ml di reagente CellTiter Glo ed incubare a temperatura ambiente per 15 min. </li><li> Dopo l&#39;incubazione, luminescenza conteggio misura dei campioni e degli standard usando un lettore di micropiastre e calibrare campione concentrazioni di ATP utilizzando una curva di regressione lineare generato dalle norme ATP. </li><li> Determinare la concentrazione totale di ATP presente nel campione e moltiplicare per il fattore di diluizione del campione per ottenere la concentrazione intracellulare di ATP in una cella carica di idrogel. </li></ol> G.3. Determinare la secrezione di insulina <ol><li> Calcolare la Amount di insulina secreta dalle sferoidi recuperati prendendo il rapporto della quantità di insulina secreta nel terreno di glucosio elevata quantità di insulina secreta nel terreno di glucosio basso. </li><li> Normalizza il contenuto di insulina quantificata sferoidi cellulari con quantità corrispondente di ATP intracellulare degli sferoidi di cellule recuperate. </li></ol>

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Il protocollo descritto presenta dettagli facile incapsulamento di cellule in tiolo-ene idrogel formati da passo-crescita fotopolimerizzazione. Mentre un rapporto stechiometrico di 1:1 di norbornene a tiolo gruppi funzionali è stato utilizzato in questo protocollo, il rapporto può essere regolata a seconda delle esperienze. Oltre a una corretta formulazione, è importante mantenere omogeneità nella pre-polimero soluzione. In particolare, utilizzare pipettaggio delicato per garantire che le cellule sono ben distribuit...

Gli idrogel sono idrofili polimeri reticolati con un potenziale eccezionale come materiali ponteggi per la riparazione e la rigenerazione dei tessuti. 1-3 L&#39;alto contenuto di acqua degli idrogel permette facile diffusione di ossigeno e di scambio di nutrienti e prodotti del metabolismo cellulare, che sono tutti fondamentale per mantenere la vitalità cellulare. Inoltre, gli idrogel sono vettori eccellenti per rilascio controllato e consegna cellulare dovuta loro sintonizzabilità alta. 2 idrogel sintetici come quelli preparati da poli (etilene glicole) (PEG) sono sempre più utilizzati in applicazioni di ingegneria tissutale, soprattutto a causa della loro citocompatibilità, tessuto- elasticità simile, e sintonizzabilità alta in materiale proprietà fisiche e meccaniche. 4-6 Sebbene una piattaforma idrogel comunemente usato, studi hanno dimostrato che PEG diacrilato (PEGDA) idrogel formato da catene crescita photopolymerizations tendono a danneggiare le cellule incapsulate durireticolazione rete ng e in situ incapsulamento delle cellule. 7 Il danno cellulare è stato largamente attribuito alla specie radicali generati dalle molecole di fotoiniziatore che si propagano attraverso i gruppi vinilici su PEGDA per reticolare catene polimeriche in idrogel. Purtroppo, queste specie radicali anche causare sollecitazioni e danno cellulare durante l&#39;incapsulamento delle cellule, in particolare per radicale cellule sensibili come β-cellule pancreatiche. 8-10 Per ottenere una dimensione di maglia superiore per una migliore diffusione e la sopravvivenza cellulare, maggiore peso molecolare PEGDA vengono spesso utilizzati per l&#39;incapsulamento delle cellule. Questo, tuttavia, compromette cinetica di polimerizzazione e provoca sub-ottimali proprietà biofisiche gel. 7,11,12 Oltre ai suddetti inconvenienti, è molto difficile recuperare strutture cellulari da idrogel PEGDA causa della natura eterogeneità e non degradabile dei le reti reticolati. Mentre proteasi sensibili peptidi possono essere incorporatinel backbone macromero PEG rendere gli idrogel altrimenti inerti PEGDA sensibili alla scissione enzimatica, la coniugazione usa spesso reagenti costosi e le reti risultanti contengono ancora un&#39;elevata eterogeneità a causa della natura della catena crescita polimerizzazione. 13-15 Recentemente, PEG-peptide idrogel formati tramite step-crescita tiolo-ene fotopolimerizzazione hanno dimostrato di esibire proprietà preferenziali per incapsulamento di cellule nel idrogel formati dalla catena crescita fotopolimerizzazione. 7 La cinetica di gelazione superiori di tiolo-ene idrogel è attribuita al &#39;click &#39;natura della reazione tra tiolo e funzionalità ene. Rispetto alla catena crescita polimerizzazione di PEGDA, tiolo-ene reazione è inibita meno ossigeno che si traduce in velocità di gelificazione veloce. 16,17 tiolo-ene idrogel hanno anche maggiore efficienza di polimerizzazione e le proprietà biofisiche gel migliori rispetto alla catena crescita PEGDA idrogel, 7 , 18 &lt;/ Sup&gt; che si traduce in limitate danni cellulari provocati dai radicali specie durante la fotopolimerizzazione. Precedentemente, tiolo-ene idrogel formato dal braccio 4-PEG-norbornene (PEG4NB) macromero e bis-cisteina reticolanti contenenti peptidi, come proteasi sensibili peptidi sono stati utilizzati per l&#39;incapsulamento delle cellule. 7,18 sintonizzabilità Massima reti idrogel PEG offre flessibile e controllabile microambiente 3D per indagare la sopravvivenza cellulare e l&#39;attività, mentre l&#39;utilizzo di proteasi sensibile sequenza peptidica fornisce un modo delicato per il recupero dei costrutti cellulari formano naturalmente all&#39;interno di idrogel. In questo protocollo utilizziamo passo-crescita photopolymerized tiolo-ene idrogel fabbricato utilizzando 4-braccio PEG-norbornene (PEG4NB) e chimotripsina sensibile reticolante peptide (CGGY ↓ C) per l&#39;incapsulamento di MIN6 β-cellule. Questo protocollo elabora sistematicamente le tecniche per lo studio della formazione di sopravvivenza, proliferazione e sferoidale di MIN6β-cellule tiolo-ene idrogel. Abbiamo inoltre fornisce metodo per β-cellule recupero sferoide e caratterizzazione biologica di sferoidi recuperati.

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> 4 bracci PEG (20kDa) </td><td> Jenkem Technology USA </td><td> 4ARM-PEG-20K </td><td></td></tr><tr><td> Fmoc-amminoacidi </td><td> Anaspec </td><td></td><td></td></tr><tr><td> Live / Dead cella kit vitalità </td><td> Invitrogen </td><td> L3224 </td><td> Include Calcein AM e etidio omodimero-1 </td></tr><tr><td> AlamarBlue reagente </td><td> ABD Serotec </td><td> BUF012 </td><td></td></tr><tr><td> CellTiter Glo reagente </td><td> Promega </td><td> G7570 </td><td></td></tr><tr><td> DPBS </td><td> Lonza </td><td> 17-512F </td><td> Senza Ca +2 e Mg +2 </td></tr><tr><td> HBSS </td><td> Lonza </td><td> 10547F </td><td> SenzaCa +2 e Mg +2 </td></tr><tr><td> Alta glucosio DMEM </td><td> Hyclone </td><td> SH30243.01 </td><td></td></tr><tr><td> FBS </td><td> Gibco </td><td> 16000-044 </td><td></td></tr><tr><td> Antibiotico-antimicotica </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> β-Mercaptoethanol </td><td> Sigma-Aldrich </td><td> M7522-100ML </td><td></td></tr><tr><td> Tripsina-EDTA </td><td> Invitrogen </td><td> 15400-054 </td><td></td></tr><tr><td> Tripsina-free α-chimotripsina </td><td> Worthington Biochemical Corp </td><td> LS001432 </td><td></td></tr><tr><td> Mouse Inusin kit ELISA </td><td> Mercodia </td><td> 10-1247-01 </td><td></td></tr><tr><td> 1 ml siringa monouso </td><td> BD Biosciences </td><td></td><td></td></tr></tbody></table>

generation and recovery of &beta;-cell spheroids from step-growth peg-peptide hydrogels

Gli idrogel sono polimeri reticolati idrofili che forniscono un microambiente tridimensionale con tessuto-come elasticità ed elevata permeabilità per coltura di cellule o tessuti terapeuticamente rilevanti. Idrogel preparati da poli (etilene glicole) (PEG) derivati ​​sono sempre più utilizzati per una varietà di applicazioni di ingegneria tissutale, in parte a causa delle loro proprietà e cytocompatible sintonizzabili. In questo protocollo, abbiamo utilizzato tiolo-ene-step crescita photopolymerizations per fabbricare PEG-peptide idrogel per incapsulare pancreatica MIN6 b-cellule. I gel sono stati formati da 4-braccio PEG-norbornene (PEG4NB) e un macromero chimotripsina sensibile reticolante peptide (CGGYC). La natura idrofila e non-fouling di PEG offre un microambiente cytocompatible per la sopravvivenza e la proliferazione cellulare in 3D, mentre l&#39;utilizzo di chimotripsina-sensitive sequenza peptidica (C GGY ↓ C, freccia indica sito di clivaggio enzimatico, mentre cisti terminaleeine residui sono stati aggiunti per tiolo-ene reticolazione) permette un rapido recupero di costruzioni cellulari che formano all&#39;interno del idrogel. Il seguente protocollo elabora tecniche per: (1) Incapsulamento di MIN6 β-cellule nel tiolo-ene idrogel, (2) analisi qualitativa e quantitativa della vitalità cellulare per determinare la sopravvivenza e la proliferazione cellulare, (3) Recupero di sferoidi cellulari usando chimotripsina-mediata gel erosione, e (4) Analisi strutturale e funzionale degli sferoidi recuperati.

<html> Figure 1-4 Mostra risultati rappresentativi per l&#39;incapsulamento, la sopravvivenza, la proliferazione, la formazione di sferoide, e il recupero sferoidale in tiolo-ene idrogel. Figura 1 mostra lo schema di reazione (1) fase di crescita tiolo-ene fotopolimerizzazione con PEG4NB e CGGYC, e ( 2) l&#39;erosione chimotripsina gel mediata che segue un meccanismo di erosione superficiale. figure 2 e 3, i risultati ottenuti con la vitalità presenti c...

Watch this Scientific Journal Video about Generation and Recovery of Î²-cell Spheroids From Step-growth PEG-peptide Hydrogels at JoVE.com

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Il seguente protocollo fornisce tecniche per l&#39;incapsulamento pancreatiche β-cellule in fase di crescita PEG-peptide idrogel formati da tiolo-ene foto-click reazioni. Questa piattaforma materiale non solo offre un microambiente cytocompatible per l&#39;incapsulamento delle cellule, ma anche controllato dall&#39;utente consente un rapido recupero delle strutture cellulari formano all&#39;interno dei idrogel.

Generazione e recupero dei β-cellule Spheroids dal passaggio di crescita PEG-peptide idrogel

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

generation-recovery-cell-spheroids-from-step-growth-peg-peptide

Research

JoVE Journal

Bioengineering

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

14.1K Views.  Indiana University - Purdue University at Indianapolis. Il seguente protocollo fornisce tecniche per l'incapsulamento pancreatiche β-cellule in fase di crescita PEG-peptide idrogel formati da tiolo-ene foto-click reazioni. Questa piattaforma materiale non solo offre un microambiente cytocompatible per l'incapsulamento delle cellule, ma anche controllato dall'utente consente un rapido recupero delle strutture cellulari formano all'interno dei idrogel.

Video: Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Un semplice Hanging goccia Cultura protocollo cellulare per la generazione di sferoidi 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Ricerca

Bioingegneria

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Idrogeli di alginato per tridimensionale Cultura Organo di ovaie e tube

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formazione Light-mediata ed il modello di idrogel per colture cellulari

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Sintonizzabile idrogel da polmonare matrice extracellulare per colture cellulari 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Produzione di elastina-come proteina idrogeli per incapsulamento e Immunostaining delle cellule in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Ceppo controllato di idrogel 3D in imaging a microscopia dal vivo

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

Biofabbricazione assistita da microscopia dell'ossigeno a prezzi accessibili di sferoidi multicellulari

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

Frammentazione di idrogel sfusi e lavorazione in idrogel granulari per applicazioni biomediche

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Sferoidi epatici umani da sangue periferico per studi sulle malattie del fegato

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Generazione, screening ad alto rendimento e biobanca di sferoidi cardiaci derivati da cellule staminali pluripotenti indotte dall'uomo

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...