The authors would like to acknowledge funding from Ministry of Education AcRF Tier 1 (RG41) and start up grant from Nanyang Technological University. Low and Tjin are funded by the Research Student Scholarship (RSS) from Nanyang Technological University, Singapore.

1. Cloning of Recombinant Plasmids Encoding for aECM Proteins
<ol>
	<li>Design the amino acid sequence of the functional domain (e.g., cell-binding domain and elastin-like repeats). Design restriction sites flanking the ends of the functional domains to facilitate sub-cloning using free software according to software instructions (e.g., http://biologylabs.utah.edu/jorgensen/wayned/ape/). Here, select unique restriction sites that are not present in the functional domain to confine digestion to the intended sites. Choose restriction sites that appear in the multiple cloning site (MCS) of the host vector (e.g., pET22b(+)) for sub-cloning.</li>
	<li>Reverse translate the amino acid sequence into the nucleotide sequence using free websites according to software instructions. (e.g., http://www.bioinformatics.org/sms2/rev_trans.html). Ensure codons are optimized for E. coli hosts.</li>
	<li>Gene of interest can be purchased commercially as single stranded oligonucleotides (i.e., if the oligonucleotides &#60; 100 base pairs (bp)) and perform DNA annealing (see step 1.4) to reduce the cost. Otherwise, for genes larger than 100 bp, they could be purchased through commercial companies.</li>
	<li>DNA annealing of the oligonucleotides
	<ol>
		<li>Anneal the DNA oligomers to obtain the desired gene sequence. Dissolve DNA oligonucleotides in a DNA oligomer buffer (10 mM Tris: 2-amino-2-(hydroxymethyl)-propan-1,3-diol, pH 8.0, filtered) to a final concentration of 1 &#181;g/&#181;l.</li>
		<li>Add 4 &#181;l of each oligomer to 32 &#181;l of DNA annealing buffer (10 mM Tris, 100 mM NaCl and 100 nM MgCl2) to achieve a total 40 &#181;l mixture.</li>
		<li>Boil a beaker of water using a hotplate and immerse the mixture for 5 min, 95 &#176;C. Remove the beaker and gradually cool the entire set up in a Styrofoam box O/N. The oligomers have been annealed and are ready for digestion.</li>
	</ol>
	</li>
	<li>Add the corresponding restriction enzymes to digest the annealed oligomers (i.e., referred to as insert) and host vector (i.e., pET22b(+)) separately. Use the following recipe (1-2 &#181;g of DNA, 2 &#181;l of each restriction enzyme, 5 &#181;l of 10x restriction enzyme buffer, and water up to a total 50 &#181;l mixture) for 3-4 hr at 37 &#176;C. 
	NOTE: The pET22b(+) plasmid vector is ampicillin antibiotic-resistant and contains a 6x-His tag at the C-terminus. The 6x-His tag present in pET22b(+) allows us to use His-tagged antibodies to identify the target protein via western blotting in step 7.</li>
	<li>Add 6x loading dye to each digestion mixture. Run the digestion mixtures separately including a DNA ladder on 1.2% DNA agarose gels containing a UV-fluorescent DNA stain for 1 hr at 100 V. Visualize the 1.2% DNA agarose gel with a UV light illuminator.</li>
	<li>Slice the gel to extract digested DNA products using commercial gel purification kits. Elute using the minimum volume for the column to achieve a minimum DNA concentration of 50-100 ng/&#181;l.</li>
	<li>Combine the gene of interest sequentially by ligating the digested DNA insert (i.e., elastin repeats or cell binding domains obtained by DNA annealing) into the plasmid vector using T4 ligase using the following recipe: (2 &#181;l of vector, 1 &#181;l of T4 ligase, 1.5 &#181;l of T4 ligation buffer, x &#181;l of insert, 10.5-x &#181;l water for a total 15 &#181;l mixture). Incubate the ligation mixture at RT&#160;for 2 hr. 
	NOTE: Molar concentration of vector to insert should be varied to optimize ligation efficiency. Volume of insert described as x in the recipe depends on the eluted DNA concentration from step 1.7.</li>
	<li>Thaw E. coli DH5&#945; chemically competent cells (or any cloning strain) on ice. Warm 2xYT agar plates (Table 1) containing ampicillin (25 &#181;g/ml) to 37 &#176;C.</li>
	<li>Transform the cells using heat shock:
	<ol>
		<li>Aliquot 50 &#181;l of competent cells into clean, pre-chilled micro-centrifuge tubes. Pipette 5 &#181;l (between 100 pg to 100 ng) of the ligation mixture into the cells, pipetting gently up and down to mix. Leave the mixture on ice for 20 min.</li>
		<li>Immerse the micro-centrifuge tube containing the cell mixture in a 42 &#176;C water bath for 2 min and returned to on ice for 2 min. Time the immersion duration to minimize heat damage to the cells.</li>
		<li>Add 500 &#181;l of SOC media (Table 1) into the micro-centrifuge tube and incubate at 37 &#176;C with shaking for 1 hr.</li>
		<li>Spread 50 500 &#181;l of the cell/ligation mixture onto a 2xYT agar plate that has been pre-warmed to RT, containing ampicillin (25 &#181;g/ml) and incubate plates upside-down at 37 &#176;C O/N (12-16 hr).</li>
	</ol>
	</li>
	<li>The next day, pick DNA colonies from the agar plate using clean pipette tips. Grow the colonies in 5 ml of 2YT media (Table 1) containing ampicillin (25 &#181;g/ml) O/N at 37 &#176;C O/N (12-16 hr) with shaking (225 rpm).</li>
	<li>The following day, use a plasmid isolation kit to extract the DNA plasmids for each colony picked according to manufacturer&#8217;s protocol. Elute the DNA with 50 &#181;l of water.</li>
	<li>Perform a test digest with restriction enzymes using the following recipe: (5 &#181;l DNA, 0.2 &#181;l of each restriction enzyme, 1 &#181;l 10x restriction enzyme buffer and top up water for a total 10 &#181;l mixture), incubate for 2 hr at 37 &#176;C to screen for colonies that likely contain the insert and run on 1.2% DNA agarose gel as in step 1.6. 
	NOTE: Upon digestion, a successful ligation should results in two bands, one each for vector and insert which correspond to their respective molecular weight (Figure 1).</li>
	<li>Send the likely colonies for DNA sequencing using T7 promoter forward and reverse primers to commercial sequencers.</li>
</ol>
2. Transformation of Recombinant Plasmid into Bacterial Expression Host
<ol>
	<li>From the sequencing result, select a colony that has been successfully ligated and use the DNA plasmid for transformation into an E. coli expression host.</li>
	<li>Thaw E. coli expression strain (BL21(DE3)pLysS) on ice. Meanwhile, warm agar plates containing ampicillin (25 &#181;g/ml) and chloramphenicol (34 &#181;g/ml)&#160;to RT. 
	NOTE: The pLysS plasmid present in bacteria strain BL21(DE3)pLysS contains a chloramphenicol resistance gene. The pLysS plasmid contains a T7 repressor gene which is constitutively expressed to limit leaky expression of the aECM protein. Chloramphenicol is necessary to select for bacteria cells that contains pLysS during culture.</li>
	<li>Repeat step 1.10 to obtain transformed E. coli cells that are ready to express the artificial proteins. Wrap agar plates in Parafilm and store upside-down in 4 &#176;C for up to one month.</li>
</ol>
3. Bacterial Expression of aECM Proteins
<ol>
	<li>Refer to Figure 2, pick a colony from the transformed agar plate with a pipette tip and inoculate into 10 ml of sterile Terrific Broth (TB) media (Table 1) containing both ampicillin and chloramphenicol antibiotics in a test tube. Incubate this starter culture at 37 &#176;C O/N (12-16 hr) with shaking at 225 rpm.</li>
	<li>Transfer 10 ml of the starter culture into 1 L fresh and sterile TB media supplemented with the same antibiotics in a 3 L Erlenmeyer flask. Incubate the culture at 37 &#176;C with shaking at 225 rpm for 2-3 hr and observe the optical density of the culture (OD600) reached 0.6-0.8 by pipetting 1 ml of culture into an empty cuvette for reading. Save 1 ml of culture prior to induction for SDS-PAGE characterization.
	<ol>
		<li>To measure OD600 of the cell culture, prepare 1 vial of TB media in a cuvette for a blank measurement. Separately, transfer 1 ml of culture from the culture flask into a new empty cuvette. Measure the optical density of the culture using a spectrophotometer against the blank control at an absorbance of 600 nm.</li>
		<li>Save the sample (for subsequent SDS-PAGE analysis) by transferring the 1 ml of culture sample into a 1.5 ml microcentrifuge tube, centrifuge at 12,000 x g for 2 min, and decant supernatant.</li>
	</ol>
	</li>
	<li>Induce the culture with isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and incubate at 37 &#176;C with shaking at 225 rpm for another 4 hr. Save 1 ml of culture at the end of induction at 4 hr for SDS-PAGE characterization.</li>
	<li>Harvest the cells by transferring the culture to 1 L centrifuge bottles and centrifuge at 12,000 x g for 30 min at 4 &#176;C. Discard the supernatant, weigh the cell pellets and resuspend in TEN buffer (1 M Tris, 0.01 M EDTA, 0.1 M NaCl, pH = 8.0) at 0.5 g/ml.</li>
</ol>
4. Lysis of Bacterial Cultures
<ol>
	<li>Freeze the re-suspended cell culture at -80 &#176;C O/N. Thaw the frozen cell culture in a water bath at RT or on ice to lyse the cells. Add 10 &#956;g/ml Deoxyribonuclease I (DNase I), 10 &#956;g/ml Ribonuclease A (RNase I), and 50 &#956;g/ml phenylmethylsulfonyl fluoride (PMSF) while thawing and homogenize the solution with slow stirring.</li>
	<li>After all resuspended cells are thawed, adjust the solution to pH 9.0 to increase the protein solubility in water12. Add 6 N NaOH drop wise with stirring on ice to achieve a homogenous consistency. Lyse by ultrasonic disruption for 20 min on ice using a 2 mm diameter flat tip, 5 sec pulse.</li>
	<li>Centrifuge the cell solution at 12,000 x g for 30 min at 4 &#176;C. Transfer the supernatant to a clean, empty bottle and store at 4 &#176;C for purification later.</li>
	<li>Meanwhile, resuspend the cell pellet again with TEN buffer and re-freeze at -80 &#176;C. To complete cell lysis, repeat freeze/thaw and sonication process up to three times. Save 20 &#181;l cell lysate for SDS-PAGE characterization.</li>
</ol>
5. Purification of aECM Proteins Using Inverse Transition Cycling
<ol>
	<li>Collate the cell lysate from step 4.3 and proceed to purify the aECM proteins using ITC, similar to that used for purification of elastin-based proteins21. Cycles will be done with the different temperatures which are 4 &#176;C (referred to as &#8220;cold&#8221;) and 37 &#176;C (referred to as &#8220;warm&#8221;) cycles respectively.</li>
	<li>Split the cell lysate into 50 ml centrifuge bottles, and centrifuge at 40,000 x g for 2 hr at 4 &#176;C. The appearance of the cell pellet should be dark brown and look runny.</li>
	<li>Remove the supernatant by pipetting to get a clean separation from the pellet. Collect the supernatant in clean centrifuge bottles and add NaCl to a final concentration of 1 M (to a maximum of 3 M). Warm the solution to 37 &#176;C for 2 hr with shaking at 225 rpm. 
	NOTE: Addition of NaCl will trigger the transition of the elastin component of the aECM causing aggregation. The supernatant will turn turbid. If the protein concentration is high enough, white foamy protein can be seen at the sides of the centrifuge bottle.</li>
	<li>Centrifuge the supernatant from step 5.3 at 40,000 x g for 2 hr at 37 &#176;C. Decant the supernatant. Crush the pellet using a metal spatula and resuspend the pellet bits in ice-cold autoclaved distilled water (50 mg/ml) with vigorous stirring O/N at 4 &#176;C using a magnetic stir bar and plate. The pellet should be completely dissolved.</li>
	<li>Repeat steps 5.2 to 5.4 for another three to five times to obtain a higher purity of the protein.</li>
	<li>After the final cycle of purification, desalt the protein solution by dialyzing it against distilled water at 4 &#176;C. Dialyze protein solution against water for 2-3 days with changes in water every 4 hr or 8 hr for O/N. Save 20 &#181;l of the purified protein for SDS-PAGE and lyophilize the rest of the purified protein and store at -80 &#176;C until further use.</li>
</ol>
6. Characterization of aECM Proteins Using SDS-PAGE Electrophoresis
<ol>
	<li>Prepare a 12% SDS-PAGE gel (if the molecular weight is large, use 8% SDS-PAGE gels for better separation). Add 2x SDS loading buffer to each sample, heat the samples for 10 min at 100 &#176;C and run samples on the gel for 1 hr at 100 V or until the protein ladder corresponding to the molecular weight of the aECM protein reaches the middle of the gel.</li>
	<li>Retrieve the gel from the SDS-PAGE set up and add Coomassie Brilliant Blue stain (Table 2) with a volume to submerge the gel for 1 hr in a Petri dish. Rinse gel in a destaining solution (Table 2) for 5 min on a rocker. Change the destaining solution, and continue to destain the gel with rocking until the background of the gel becomes clear. Protein bands should be clearly visible.</li>
	<li>Compare the position of the target protein against the protein ladder to determine the molecular weight of the target protein.</li>
	<li>To further confirm the presence of the target protein, perform western blotting as in step 7.</li>
</ol>
7. Characterization of aECM Proteins Using Western Blotting
<ol>
	<li>Run the samples on a 12% SDS-PAGE gel as in section 6 with no staining.</li>
	<li>Cut nitrocellulose membrane to a size slightly larger than the SDS-PAGE gel. Cut 4 pieces of filter paper to the same size.</li>
	<li>Wet one piece of filter paper with Western Transfer Buffer (20% v/v methanol, 25 mM Tris, 190 mM Glycine, pH 8.3), place the SDS-PAGE gel on top of the filter paper, followed by another piece of filter paper, then the nitrocellulose membrane and a final other two pieces of filter paper. Ensure the whole set-up is submerged with sufficient western transfer buffer. 
	NOTE: The gel and membrane should not be in contact at this point of time as proteins could bind to the membrane upon contact.</li>
	<li>Transfer the proteins onto the nitrocellulose membrane by placing two filter papers into a western semi-dry transfer unit, followed by the nitrocellulose membrane, and carefully place the SDS-PAGE gel within and on the membrane, lastly place the last two wet filter paper on the SDS-PAGE gel. Run the western transfer at 45 mA, 30 min. Add buffer if voltage is higher than 30 V. 
	NOTE: Preferably place the SDS-PAGE gel on the membrane with one attempt and avoid shifting the gel unnecessarily on the membrane as proteins could bind to the membrane upon contact.</li>
	<li>Retrieve and block the nitrocellulose membrane with blocking solution (5% non-fat milk in PBS pH 7.4, filtered with filter paper) for 2 hr at RT. Dispose of blocking buffer and add PBS to rinse. 
	NOTE: Change to new gloves to avoid contaminating the membrane with unwanted proteins.</li>
	<li>Incubate primary anti-His antibody with dilution in PBS at 1:1,000 at RT for 1 hr with rocking. Prepare a mixture in a volume which could submerge the whole membrane, for example, with dilution ratio 1:1,000, add 1 &#181;l of antibody (stock concentration: 1 mg/ml) to 999 &#181;l PBS for a 1 ml total mixture.</li>
	<li>Rinse the membrane with 5 ml of PBST (PBS with 0.1% Tween20).</li>
	<li>Incubate with secondary antibody conjugated to horse-radish peroxidase (HRP) with dilution in PBS at 1:5,000 at RT for 1 hr with rocking. Prepare a mixture in a volume which could submerge the whole membrane, for example, with dilution ratio 1:5,000, add 1 &#181;l of antibody (stock concentration: 1 mg/ml) to 4,999 &#181;l PBS for a 5 ml total mixture.</li>
	<li>Wash the membrane with 5 ml of PBST and repeat twice.</li>
	<li>Apply the chemiluminescent substrate to the membrane with a volume to cover the membrane and incubate by following the instructions for the substrate used. 
	NOTE: The substrate&#8217;s sensitivity to protein detection may vary, we recommend a substrate with maximum sensitivity for very low signals in the event increasing antibody concentration does not increase signals.</li>
	<li>Capture the chemiluminescent signals using a CCD camera based imager. Adjust the primary and secondary antibody dilution ratio if signals are weak and non-specific. Incubate longer in blocking solution if background signal is high.</li>
</ol>

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The authors declare that they have no competing financial interests.

Recombinant protein engineering is a versatile technique to create novel protein materials using a bottom-up approach. The protein-based materials can be designed to have multiple functionalities, tailored according to the application of interest. Due to increasing advancement in cloning and protein expression technologies, it has become relatively simple (and cost effective) to create a variety of artificial proteins in a reproducible and scalable manner. The elastin-like domain has been incorporated in a number of arti...

For several decades, both synthetic and natural materials have been explored for use as scaffolds in tissue engineering1,2. While synthetic materials such as polymers offer excellent structural integrity and tunable mechanical properties, they often have insufficient bioactivity to promote growth and infiltration of tissues. On the other hand, natural materials such as extracellular matrix (ECM) proteins have excellent biological activity, but have limitations such as batch-to-batch variability, rapid degradation and immunogenicity issues. As such, recombinant proteins are desired, since they can be designed to mimic only the desirable properties of native proteins3,4.
Recombinant protein engineering has garnered widespread interests as a versatile platform for the design and production of novel artificial protein biopolymers. By controlling the genetic sequence, the functionalities of the artificial proteins can be tailored for a wide variety of applications5,6. In particular, artificial extracellular matrix (aECM) proteins can be tailored to have multiple functionalities for applications in tissue engineering, regeneration and wound repair2,7. More importantly, advances in cloning and purification technologies have increased scalability and reduced the cost of manufacturing recombinant proteins tremendously. It is possible to produce large quantities of recombinant proteins at low production costs which are economic for use in the clinic5. 
Artificial extracellular matrix proteins have been developed for tissue engineering applications8-11. For instance, Tirrell et al. designed a small diameter vascular graft using artificial proteins containing fibronectin CS5 sequence and elastin-like repeats (ELP-CS5). They showed that human umbilical vein endothelial cells (HUVECs) were able to adhere and grow on these materials12. Others have also incorporated short bioactive sequences taken from fibronectin, collagen, laminin, fibrinogen and vitronectin as well as structural domains that mimic elastin, spider silk and collagens to create a variety of fusion proteins10. Bulk cross-linked films made out of elastin-based aECM proteins also exhibited mechanical properties similar to that of native elastin (elastic moduli ranges between 0.3-0.6 MPa)13. Subsequently, aECM proteins containing longer fibronectin fragments were also reported to accelerate wound healing in vitro due to increased integrin binding affinities8. 
Recently, integrin-specific artificial ECM proteins have been developed by Tjin and coworkers14. Each aECM protein contains a bioactive cell-binding domain taken from ECM components of native human skin2,7,15, such as laminin-5, collagen-IV and fibronectin. For example, the integrin &#945;3&#914;1 has been shown to bind the PPFLMLLKGSTR sequence found in the laminin-5 alpha-3 chain globular domain 3 (LG3)16,17. In their report, they showed that primary human skin epidermal keratinocytes preferentially engage different integrins for binding to each of the aECM proteins, depending on the type of cell binding domain present.
The aECM proteins discussed in the work by Tjin et al. contain flanking elastin-like domains {(VPGIG)2VPGKG(VPGIG)2}8 that confer elasticity which mimics the mechanical properties of human skin. In addition, the incorporation of lysine residues within the elastin-like repeats also increases the overall protein solubility in aqueous solvents. In addition, the lysine residues also serve as crosslinking sites to facilitate the formation of crosslinked aECM films12. Inclusion of elastin-like repeats within the aECM protein sequence allow the proteins to be readily purified via Inverse Transition Cycling (ITC)14. Elastins undergo a sharp and reversible phase transition at a specific temperature known as the lower critical solution temperature (LCST) or the inverse transition temperature (Tt)18-20. Elastins and elastin-like repeats adopt hydrophilic random coil conformations below their LCST and become soluble in water, whereas above their LCST, elastins aggregate rapidly into micron-size particles. Such phase transitions are reversible and hence, can be exploited to allow elastin-based aECM proteins to be readily purified via the ITC technique21.
In this work, we report a generalized procedure to design, construct and purify artificial ECM proteins containing bioactive cell-binding domains, fused to elastin-like repeats. The process to design and clone the plasmids that encode for the amino acid sequences for the aECM proteins is described. The steps involved to purify the aECM proteins using ITC are outlined. Finally, the methods to determine the purity of the aECM proteins obtained using SDS-PAGE electrophoresis and Western Blotting are discussed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>pET22b (+)</td>
			<td>Novagen</td>
			<td>69744</td>
			<td>T7 expression vectors with resistance to ampicillin&#160;</td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS&#160;</td>
			<td>Invitrogen</td>
			<td>C6060-03</td>
			<td>additional antibiotics - chloramphenicol</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside (IPTG)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C</td>
			<td>1M stock solution with autoclaved water, make fresh prior to induction.</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>plasmid isolation kit</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Affymetrix</td>
			<td>23660</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymoclean&#8482; gel DNA recovery kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-gold strain</td>
			<td>Agilent Technologies</td>
			<td>200315</td>
			<td></td>
		</tr>
	</tbody>
</table>

design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

In designing fusion proteins containing elastin-like repeats, it is important to maintain an overall elastin content, large enough fraction of the fusion protein18. This is to ensure that the fusion protein construct retains its elastin-like characteristics, in order to use ITC for purification. The aECM proteins design and sequences described in this section were specifically taken from the work by Tjin et al.14. In this work, three aECM proteins were successfully cloned into the ...

Watch this Scientific Journal Video about Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering at JoVE.com

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

design-construction-artificial-extracellular-matrix-aecm-proteins

Research

JoVE Journal

Bioengineering

8.8K Views.  Nanyang Technological University. Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats. 

Video: Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

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.

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

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

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.

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

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...