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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

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

Published: May 19th, 2018

DOI:

10.3791/57739

1Department of Bioengineering, Stanford University, 2Department of Materials Science and Engineering, Stanford University
* These authors contributed equally

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.

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.

Over the past century, two-dimensional (2D) tissue culture has developed into an integral toolset for studying fundamental cell biology in vitro. In addition, the relatively low-cost and simple protocols for 2D cell culture have led to its adoption across many biological and medical disciplines. However, past research has shown that traditional 2D platforms can lead to results that deviate markedly from those collected in vivo, causing precious time and funding wasted for clinically oriented research1,2,3. We and others hypothesize that this discrepancy may ....

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1. ELP Expression Protocol

  1. Day 1: Growing the starter colony
    1. Prepare ampicillin and chloramphenicol agar plates by autoclaving 25 g of Luria broth and 15 g of agar per 1 L of ultrapure water. Once the solution has cooled to ~60 °C, add 1 mL of ampicillin stock (100 mg/mL in ultrapure water) and 1 mL of chloramphenicol stock (34 mg/mL in 70% ethanol) to 1 L of agar solution for final concentrations of 100 µg/mL and 34 µg/mL, respectively. Transfer 20 mL of final solu.......

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The ELPs used in this protocol are comprised of five regions: a T7 tag, His6 tag, enterokinase (EK) cleavage site, a bio-active region, and an elastin-like region (Figure 1). The T7 and His6 tags allow for easy identification through standard Western blot techniques. Introduction of the EK cleavage site allows for the enzymatic removal of the tag region, if needed. The bio-active region encodes for the extended, fibronectin-derived cell-adhesive ('RGDS.......

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Recombinant protein expression and purification is a powerful tool to synthesize biomaterials with high reproducibility. Owing largely to the advent of commercialized molecular cloning, custom recombinant plasmids can be purchased from several suppliers, which significantly reduces the time to work with materials like ELPs. Similarly, plasmids can be requested directly from the originating lab when the original work was supported by a federal contract and the future work will be for non-profit use. The full ELP amino aci.......

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The authors thank T. Palmer and H. Babu (Stanford Neurosurgery) for providing murine NPCs. Vector art in Figure 4 was used and adapted from Servier Medical Art under Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/legalcode). Part of this work was performed at the Stanford Nano Shared Facilities (SNSF), supported by the National Science Foundation under award ECCS-1542152. N.A.S. acknowledges support from the National Institute of General Medical Sciences of the National Institutes of Health (32GM008412). C.M.M. acknowledges support from an NIH NRSA pre-doctoral fellowship (F31 EB020502) and ....

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NameCompanyCatalog NumberComments
Elastin-Like Protein Expression and Purification
10 cm Petri DishesThermo Fisher ScientificFB0875713
70% EthanolRICCA Chemical2546.70-1
Ammonium SulfateSigma-AldrichA3920-500G
AmpicillinThermo Fisher ScientificBP1760-25G
Bacto AgarThermo Fisher Scientific9002-18-0 
BL21(DE3)pLysS Competent CellsInvitrogenC606003
ChloramphenicolAmresco0230-100G 
Deoxyribonuclease I from bovine pancreasSigma-AldrichDN25
EDTA disodium salt, dihydrateThermo Fisher ScientificO2793-500
GlycerolThermo Fisher ScientificBP229-4
IsopropanolThermo Fisher ScientificA451-4
Isopropyl β-D-1-thiogalactopyranoside (IPTG) Thermo Fisher ScientificBP1755-10G
Luria BrothEMD Millipore1.10285.5007
ParafilmVWR52858-000
Phenylmethanesulfonyl fluoride (PMSF)MP Biomedicals195381
Sodium ChlorideThermo Fisher ScientificBP358-212
Sodium HydroxideSigma-AldrichS 8045-1KG
Syringe Filter Unit (0.22 μm)MilliporeSLGP033RB
Terrific BrothMillipore71754-4
Tris BaseThermo Fisher ScientificBP152-1
Cell Encapsulation in 3D ELP Hydrogels
0.22 μm syringe filtersMilliporeSLGV004SL
0.5 mm thick silicone sheetElectron Microscopy Science70338-05
24-well tissue culture plates Corning353047
Disposable Biopsy Punch (2 mm)Integra Miltex33-31
Disposable Biopsy Punch (4 mm)Integra Miltex33-34
Disposable Biopsy Punch (5 mm)Integra Miltex33-35
Dulbecco’s phosphate buffered saline (DPBS) Corning21-031-CM
No. 1 12 mm glass coverslipsThermo Fisher Scientific12-545-80
Tetrakis(hydroxymethyl)phosphonium chloride (THPC)Sigma-Aldrich404861-100ML
0.5% Tryspin/EDTAThermo Fisher 15400054
Immunocytochemistry of Cells in 3D ELP Hydrogels
16% (w/v) Paraformaldehyde (PFA)Electron Microscopy Sciences15701
Bovine Serum Albumin (BSA)Roche3116956001
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)Molecular ProbesD1306 
Donkey SerumLampire Biological Labs7332100
Goat anti-mouse Secondary Antibody (AF488)Molecular ProbesA-11017
Goat anti-rabbit Secondary Antibody (AF546)Molecular ProbesA-11071
Goat SerumGibco16210-072
Mouse Nestin Primary AntibodyBD Pharmingen556309
Mouse Sox2 Primary AntibodyCell Signaling Technology23064S
Nail PolishElectron Microscopy Sciences72180
Triton X-100Sigma-AldrichX100-100ML
Vectashield Hardset Mounting Medium Vector LabsH-1400 

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