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><tbody><tr><td>pET22b (+)</td><td>Novagen</td><td>69744</td><td>T7 expression vectors with resistance to ampicillin&amp;nbsp;</td></tr><tr><td>BL21(DE3)pLysS&amp;nbsp;</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>1 M 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&amp;trade; 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 ...

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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

Universidad de Cartagena UDEC

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

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 ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Developmental Biology

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

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, ...

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

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

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

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

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Efficient 3D Skin Models for Flexible Research Application

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

Author Spotlight: Enhancing Skin Model Diversity with Cost-Effective 3D Cellular Models

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

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

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

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

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

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Generation of Self-assembled Vascularized Human Skin Equivalents

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture