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

Summary

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.

Abstract

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.

Introduction

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 research^1,2,3. We and others hypothesize that this discrepancy may ....

Protocol

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 solution to 10 cm Petri dishes with a serological pipette and allow the agar to solidify. Wrap the Petri dishes with parafilm and store at 4 °C.

Results

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') or n.......

Discussion

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.......

Materials

List of materials used in this article

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