Die Autoren danken T. Palmer und H. Babu (Stanford Neurochirurgie) für die Bereitstellung von murinen NPCs. Vektorgrafiken in Abbildung 4 wurde eingesetzt und angepasst von Servier Medical Art unter Creative Commons Attribution 3.0 Unported Lizenz (https://creativecommons.org/ licenses/by/3.0/legalcode). Teil dieser Arbeit wurde an der Stanford Nano geteilt Einrichtungen (SNF), unterstützt von der National Science Foundation unter Award ECCS-1542152 durchgeführt. N.A.S erkennt Unterstützung aus dem National Institute of General Medical Sciences von den National Institutes of Health (32GM 008412). C.M.M. würdigt Unterstützung durch ein NIH NRSA Pre Promotionsstipendium (F31 EB020502) und Siebel Scholars Program. S.C.H. räumt Unterstützung von den National Institutes of Health (U19-AI116484 und R21 EB018407), National Science Foundation (DMR-1508006) und am California Institute for Regenerative Medicine (RT3-07948). Diese Forschung finanziell unterstützt von der Allianz für Regenerative Rehabilitation Research & Training (AR3T), die von Eunice Kennedy Shriver National Institute of Child Health und menschlichen Entwicklung (NICHD), National Institute of unterstützt wird Neurologische Erkrankungen und Schlaganfall (NINDS) und National Institute of Biomedical Imaging and Bioengineering (NIBIB) von den National Institutes of Health unter Nummer P2CHD086843 Award. Der Inhalt ist ausschließlich in der Verantwortung der Autoren und nicht notwendigerweise die Ansichten von den National Institutes of Health.

<ol>
	<li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+Cell-Matrix+Adhesions+to+the+Third+Dimension.">Taking Cell-Matrix Adhesions to the Third Dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li><li>Birgersdotter, A., Sandberg, R., Ernberg, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+perturbation+in+vitro-A+growing+case+for+three-dimensional+(3D)+culture+systems.">Gene expression perturbation in vitro-A growing case for three-dimensional (3D) culture systems.</a> Seminars in Cancer Biology. 15 (5), 405-412 (2005).</li><li>G&#243;mez-Lech&#243;n, M. 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Rekombinante Proteinexpression und Reinigung ist ein leistungsfähiges Werkzeug, Biomaterialien mit hoher Reproduzierbarkeit zu synthetisieren. Aufgrund weitgehend das Aufkommen der kommerzialisierten molekularen Klonen, können benutzerdefinierte rekombinante Plasmide von mehreren Lieferanten gekauft werden die reduziert die Zeit zum Arbeiten mit Materialien wie ELPs. In ähnlicher Weise können Plasmide direkt aus dem ursprünglichen Labor angefordert werden, wenn das ursprüngliche Werk durch einen Bundesvertrag unter...

Im vergangenen Jahrhundert entwickelte zweidimensionale (2D) Gewebekultur zu einer integralen Toolset für die grundlegende Zelle Biologie in-vitro-Untersuchung. Darüber hinaus haben die relativ kostengünstige und einfache Protokolle für 2D Zellkultur zu ihrem Erlass in vielen biologischen und medizinischen Disziplinen geführt. Jedoch hat Forschung gezeigt, dass traditionelle 2D Plattformen zu Ergebnissen führen können, dass deutlich von diesen gesammelten in Vivoabweichen, wodurch wertvolle Zeit und Mittel für klinisch orientierte Forschung1,2verschwendet, 3. Wir und andere stellen die Hypothese auf, dass diese Diskrepanz zugeschrieben werden kann, auf den Mangel an einheimischen biochemischen und biophysikalischen Hinweise bereitgestellt, um die Zellen kultiviert auf 2D Oberflächen, die für optimale Vermehrung und Reifung der verschiedenen Zelltypen erforderlich sein können.
Um diese Einschränkungen und Hilfe Brücke die Lücke zwischen 2D anzugehen haben in Vitro und in Vivo Studien, Forscher entwickelten dreidimensionalen (3D) Hydrogel Plattformen für Zelle-Kapselung1,4,5 ,6. Hydrogele sind ideale Materialien zu rekapitulieren die endogenen Mikroumgebung von der extrazellulären Matrix (ECM) in Vivo aufgrund ihrer Gewebe-ähnliche mechanische Eigenschaften und Wasser geschwollen Struktur, schnellen Transport von Nährstoffen und Signalisierung Faktoren7,8. Darüber hinaus können 3D Hydrogele sollen unabhängige Kontrolle der mechanischen und biochemischen Eigenschaften des Gerüstes zu haben. Matrix Mechanik9,10,11,12 und Zelle-Klebstoff Liganden13,14,15 sind bekannte Zelle beeinflussen Verhalten in Vitro und in-vivo. So bieten 3D Hydrogele mit einstellbaren Eigenschaften eine Plattform, um kausale Zusammenhänge zwischen Zellen und ihrer Mikroumgebung zu studieren. Kriterien für eine ideale 3D hydrogelmatrix sind einfache, nicht-zytotoxische Zelle-Kapselung sowie unabhängige Einstellbarkeit der physiologisch relevanten mechanischen Eigenschaften und Mimik native Zelle-Klebstoff-Motive.
Beide synthetischen (zB., Polyethylenglykol, Polymilchsäure, Poly (glykolische Säure)) und natürlich gewonnen (zB., Alginat, Kollagen, Matrigel) Hydrogele haben Vorteile gegenüber 2D in-vitro- Kultur-Plattformen; Sie haben jedoch auch erhebliche Mängel, die ihre Anwendbarkeit zu begrenzen. Erstens viele synthetische und natürlich gewonnenen Plattformen erfordern harte Vernetzung Bedingungen, die für Säugerzellen potenziell giftig sein können, führt zu verringerte Zelle Lebensfähigkeit7. Darüber hinaus viele synthetische Plattformen fehlt native Bioaktivität und müssen durch chemische Nebenreaktionen funktionalisiert werden die erhöhten Kosten und Komplexität16hinzufügen können. Schließlich, während natürlich gewonnenen Materialien in der Regel innere bioaktive Domänen enthalten, sie sind oft durch hohe Variabilität von Charge zu Charge geplagt und beschränken sich oft auf Bildung relativ schwachen Gele7,17.
Rekombinantes Protein-Engineering präsentiert eine einzigartige Toolset für Materialien Design durch explizite Kontrolle über Proteinreihenfolge und, durch Verlängerung, die möglichen mechanischen und biochemischen Eigenschaften der endgültige Hydrogel Gerüst18. Darüber hinaus können durch die Nutzung der bekannten biologischen Maschinen von Escherichia coli (E. Coli), Proteine zu äußern, Materialien kostengünstig und konsequent mit begrenzten inter - und Intra-Batch Variabilität produziert werden. Das Elastin-wie Protein (ELP) hier vorgestellten hat drei technische Bereiche: (1) ein T7 und His6 Tag, das ermöglicht eine Kennzeichnung über Gewebekulturen Antikörper markiert, (2) ein "Elastin-Like"-Region, das verleiht elastische mechanische Eigenschaften und chemische ermöglicht Vernetzung, und (3) eine "Bio-aktiv"-Region, die für Zelle-Klebstoff Motive kodiert.
Elastin-ähnliche Region basiert auf der kanonischen (Val-Pro-Gly-Xaa-Gly)5 Elastin Sequenz, wo vier 'Xaa' Aminosäure-Websites sind Isoleucin (Ile), aber könnte so konzipiert, dass jede Aminosäure außer Prolin. Diese Sequenz stiftet rekombinante ELPs mit untere kritische Temperatur (LCST) lösungsverhalten, die für einfache Reinigung nach dem Ausdruck über thermische Radfahren19,20ausgenutzt werden können. Diese LCST Eigenschaft kann bei unterschiedlichen Temperaturen durch Ändern der Gast 'Xaa' Rückstand21,22, thermisch Aggregat abgestimmt werden.
Hier wurde mit Amin-präsentierenden Lysin (Lys) Aminosäure, die "Xaa" Position auf einem der fünf Elastin-artigen Wiederholungen ersetzt, die Hydrogel Vernetzung genutzt wird. Unsere bisherige Arbeit hat nicht zytotoxisch und robuste Vernetzung durch Reaktion mit der Amin-reaktive Vernetzer Tetrakis (Hydroxymethyl) Phosphonium-Chlorid (THPC)23gezeigt. Durch unterschiedliche allgemeine Inhalte und Vernetzer Proteinkonzentration können wir Hydrogele zu produzieren, die optimiert werden können, um eine physiologisch relevanten Steifigkeit Palette (~0.5-50 kPa)9,23,24zu überspannen. Neben der Optimierung der mechanischer Eigenschaften, Zelle Adhäsion innerhalb der Hydrogel-Ergebnisse aus der Integration der kanonischen Zelle-Klebstoff-Domänen innerhalb das Rückgrat des ELP-Proteins. Beispielsweise ermöglicht die Einbeziehung der erweiterten Fibronektin abgeleitet "RGDS" Aminosäure-Sequenz Zelladhäsion und Konformationsänderungen Flexibilität, während das Rührei, unverbindliche "RDGS" Variante schränkt Zellmatrix Haftung24. Durch die Modulation des Verhältnis der Zelle-Klebstoff nichtklebenden Proteine sowie die Gesamt-Protein-Konzentration, können wir effektiv Hydrogele zu produzieren, die eine Vielzahl von Liganden-Konzentration zu überspannen. Resultantly, entwickelten wir eine Hydrogel-Plattform mit entkoppelten biochemischen und biophysikalischen Eigenschaften, die unabhängig für optimale 3D Culture von verschiedenen Zelltypen abgestimmt werden können.
Neben Matrix Steifigkeit und Klebstoff Liganden Einstellbarkeit bieten rekombinante Hydrogele die Möglichkeit, bestimmte Materialabbau designprofile, die notwendig ist für die Zelle verbreiten, Proliferation und Migration innerhalb einer 3D Rahmen4 , 9. dieser Abbau wird durch Zelle Sekretion von Proteasen, die gezielt die erweiterte "RGDS"9 oder Elastin-Sequenz25gewährt. ELP Hydrogele haben auch gezeigt, dass die nachfolgenden biochemischen Assays zu unterstützen, die notwendig sind für das Studium Zellviabilität und Immunocytochemistry, sowie DNA/RNA/Proteingewinnung für quantitative Reverse-Funktion Transkription-Polymerase Chain Reaction (qRT-PCR) und Western blot9. ELP-Varianten wurden auch in einer Reihe von in Vivo Modellen verwendet und sind dafür bekannt, die vom Immunsystem26gut vertragen werden.
Zusammengenommen, ELP als eine wesentliche Plattform für Zelle-Kapselung Studien verfügt über eine Vielzahl von Vorteilen im Vergleich zu synthetischen oder natürlich gewonnenen Material Plattformen, die oft nicht über den gleichen Grad an biochemischen und biophysikalischen Einstellbarkeit und Reproduzierbarkeit. Darüber hinaus ELP einfach und nicht zytotoxischen Einsatz mit einer Vielzahl von Zelltypen (zB., Küken Dorsal Root Ganglien14,24, murine neuronalen Vorläuferzellen Zellen9, menschlichen mesenchymalen Stammzellen27, bovine neonatale Chondrozyten28, Human endothelial Zellen29,30) ermöglicht eine mehr physiologisch relevanten Modell des endogenen 3D ECM im Vergleich zu 2D Zellkultur. Hier präsentieren wir Ihnen ein Protokoll für den Ausdruck von rekombinant abgeleitet, ELPs für den Einsatz als Durchstimmbare Hydrogel Plattform für 3D Handy Kapselung. Weiter stellen wir die Methodik für die Downstream-fluoreszierende Kennzeichnung und konfokalen Mikroskopie von verkapselten Zellen.

<table><tbody><tr><td>&lt;strong&gt;Elastin-Like Protein Expression and Purification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 cm Petri Dishes</td><td>Thermo Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>70% Ethanol</td><td>RICCA Chemical</td><td>2546.70-1</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>Sigma-Aldrich</td><td>A3920-500G</td><td></td></tr><tr><td>Ampicillin</td><td>Thermo Fisher Scientific</td><td>BP1760-25G</td><td></td></tr><tr><td>Bacto Agar</td><td>Thermo Fisher Scientific</td><td>9002-18-0&amp;nbsp;</td><td></td></tr><tr><td>BL21(DE3)pLysS Competent Cells</td><td>Invitrogen</td><td>C606003</td><td></td></tr><tr><td>Chloramphenicol</td><td>Amresco</td><td>0230-100G&amp;nbsp;</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>EDTA disodium salt, dihydrate</td><td>Thermo Fisher Scientific</td><td>O2793-500</td><td></td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific</td><td>BP229-4</td><td></td></tr><tr><td>Isopropanol</td><td>Thermo Fisher Scientific</td><td>A451-4</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>BP1755-10G</td><td></td></tr><tr><td>Luria Broth</td><td>EMD Millipore</td><td>1.10285.5007</td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-000</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>MP Biomedicals</td><td>195381</td><td></td></tr><tr><td>Sodium Chloride</td><td>Thermo Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S 8045-1KG</td><td></td></tr><tr><td>Syringe Filter Unit (0.22 &amp;mu;m)</td><td>Millipore</td><td>SLGP033RB</td><td></td></tr><tr><td>Terrific Broth</td><td>Millipore</td><td>71754-4</td><td></td></tr><tr><td>Tris Base</td><td>Thermo Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>&lt;strong&gt;Cell Encapsulation in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;mu;m syringe filters</td><td>Millipore</td><td>SLGV004SL</td><td></td></tr><tr><td>0.5 mm thick silicone sheet</td><td>Electron Microscopy Science</td><td>70338-05</td><td></td></tr><tr><td>24-well tissue culture plates&amp;nbsp;</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>Disposable Biopsy Punch (2 mm)</td><td>Integra Miltex</td><td>33-31</td><td></td></tr><tr><td>Disposable Biopsy Punch (4 mm)</td><td>Integra Miltex</td><td>33-34</td><td></td></tr><tr><td>Disposable Biopsy Punch (5 mm)</td><td>Integra Miltex</td><td>33-35</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS)&amp;nbsp;</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>No. 1 12 mm glass coverslips</td><td>Thermo Fisher Scientific</td><td>12-545-80</td><td></td></tr><tr><td>Tetrakis(hydroxymethyl)phosphonium chloride (THPC)</td><td>Sigma-Aldrich</td><td>404861-100ML</td><td></td></tr><tr><td>0.5% Tryspin/EDTA</td><td>Thermo Fisher&amp;nbsp;</td><td>15400054</td><td></td></tr><tr><td>&lt;strong&gt;Immunocytochemistry of Cells in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>16% (w/v) Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15701</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Roche</td><td>3116956001</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Molecular Probes</td><td>D1306&amp;nbsp;</td><td></td></tr><tr><td>Donkey Serum</td><td>Lampire Biological Labs</td><td>7332100</td><td></td></tr><tr><td>Goat anti-mouse Secondary Antibody (AF488)</td><td>Molecular Probes</td><td>A-11017</td><td></td></tr><tr><td>Goat anti-rabbit Secondary Antibody (AF546)</td><td>Molecular Probes</td><td>A-11071</td><td></td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210-072</td><td></td></tr><tr><td>Mouse Nestin Primary Antibody</td><td>BD Pharmingen</td><td>556309</td><td></td></tr><tr><td>Mouse Sox2 Primary Antibody</td><td>Cell Signaling Technology</td><td>23064S</td><td></td></tr><tr><td>Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>Vectashield Hardset Mounting Medium&amp;nbsp;</td><td>Vector Labs</td><td>H-1400&amp;nbsp;</td><td></td></tr></tbody></table>

production of elastin-like protein hydrogels for encapsulation and immunostaining of cells in 3d

Zweidimensionale (2D) Gewebekultur Techniken sind wichtig für unser Verständnis der grundlegenden Zelle Biologie gewesen. Jedoch sammelte traditionelle 2D Gewebekultur Systeme fehlen eine dreidimensionale (3D) Matrix, wodurch eine deutliche Trennung zwischen Ergebnisse in Vitro und in Vivo. Um diese Einschränkung zu beheben, haben Forscher 3D Hydrogel Gewebekultur Plattformen entwickelt, die die biochemischen und biophysikalischen Eigenschaften der in Vivo Zelle Mikroumgebung nachahmen kann. Diese Forschung hat die Notwendigkeit, Material Plattformen entwickeln, die 3D Zelle Kapselung und nachgelagerten biochemischen Assays unterstützen motiviert. Rekombinantes Protein-Engineering bietet eine einzigartige Toolset für 3D Hydrogel-Material-Design und Entwicklung, indem Sie für die spezifische Steuerung der Proteinreihenfolge und daher, durch Verlängerung, die möglichen mechanischen und biochemischen Eigenschaften der resultierenden Matrix. Hier präsentieren wir Ihnen ein Protokoll für die Expression von rekombinant abgeleitet Elastin-Like Protein (ELP), die Form Hydrogele mit unabhängig einstellbaren mechanischen Eigenschaften und Zelle-Klebstoff-Liganden-Konzentration genutzt werden können. Weiter präsentieren wir eine Methodik für die Zelle Kapselung in ELP Hydrogele und anschließende immunofluorescent Färbung der Zellen für nachgelagerte Analyse und Quantifizierung eingebettet.

Die in diesem Protokoll verwendeten ELPs bestehen aus fünf Regionen: ein T7-Tag, His6 Tag spaltstelle Enterokinase (EK), einer Bio-aktiven Region und ein Elastin-ähnliche Region (Abbildung 1). Die T7 und His6 Tags ermöglichen einfache Identifizierung durch standard-Western-Blot-Techniken. Einführung der EK-spaltstelle ermöglicht für den enzymatischen Abbau des Rayons Tag bei Bedarf. Die Bio-aktiven Region kodiert für die erweiterte, Fibronektin-abgelei...

Watch this Scientific Journal Video about Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D at JoVE.com

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

Rekombinantes Protein-Engineering Hydrogele sind für 3D Zellkultur von Vorteil, da sie für komplette Einstellbarkeit der Polymer-Rückgrat und sich somit die Zelle Mikroumgebung ermöglichen. Hier beschreiben wir den Prozess der rekombinante Elastin-wie Proteinreinigung und ihre Anwendung in 3D Hydrogel Zelle Kapselung.

Produktion von Elastin-Like Protein Hydrogele für Kapselung und Immunostaining Zellen in 3D

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

production-elastin-like-protein-hydrogels-for-encapsulation

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.3K Aufrufe.  Stanford University. Rekombinantes Protein-Engineering Hydrogele sind für 3D Zellkultur von Vorteil, da sie für komplette Einstellbarkeit der Polymer-Rückgrat und sich somit die Zelle Mikroumgebung ermöglichen. Hier beschreiben wir den Prozess der rekombinante Elastin-wie Proteinreinigung und ihre Anwendung in 3D Hydrogel Zelle Kapselung.

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

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Einfache Handhabung von Architekturen in Protein-basierte Hydrogele für Zelle Kultur Anwendungen

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Forschung

Biochemie

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

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

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

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Erzeugung und Verwertung von β-Zellen Spheroids Von Step-Wachstum PEG-Peptid-Hydrogele

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

Biotechnik

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

Vorbereitung der Chitosan-basierten injizierbaren Hydrogele und deren Anwendung in 3D Zellkultur

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Chemie

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Cellular Encapsulation in 3D Hydrogele für Tissue Engineering

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Biologie

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Herstellung von 3D-Kollagen-Gel und Mikrokanäle für das Studium der 3D-Interaktionen<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Technik Dreidimensionale Epithelgewebe Eingebettet in extrazellulären Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

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.

Tunable Hydrogele aus Pulmonary Extrazellulärmatrix für 3D-Zellkultur

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

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues für injizierbare Regenerative Therapie und Hochdurchsatz-Drogen-Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

Agarose-Flüssiggele, die durch Scherverarbeitung während der Gelierung für suspendierten 3D-Biodruck gebildet werden

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...

Photoabstimmbare biogedruckte Hydrogele zur Untersuchung zellulärer Reaktionen auf ECM-Versteifung

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Autor im Rampenlicht: Chronische Lungenerkrankungen mit 3D-gedruckten phototunablen Hydrogelen verstehen 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

3D-Bioprinting von phototunbaren Hydrogelen zur Untersuchung der Fibroblastenaktivierung

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...