Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.
This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.
1. Pre-processing Tissue Preparation
2. Preparation of Injectable ECM
3. Myocardial Injections
4. Representative Results
Previous characterization of matrix materials prepared in this way demonstrated the retention of a variety of macromolecules. Specifically, multiple fibrous proteins and glycoproteins were identified via mass spectrometry (Table 1). If the matrix materials processed according to this protocol are found to no longer contain a complex array of macromolecules, it could be that the decellularization protocol employed is too harsh. Once fully lyophilized, the dried pericardial ECM looks like very stiff, crumpled paper. Other tissue types will resemble packing peanuts. If milled well, the ECM should fall through the sieve and be collected in the jar at the bottom. If there is moisture left in the sample, the ECM will clump together and get stuck in the milling chamber. After digestion, the ECM solution should be a milky color and completely free of visible particulates. It will also be slightly more viscous than the HCl alone. If the ECM does not digest well, there will still be large particulates in the vial; alternatively, the ECM may crash out of solution. After pH adjustment, there is no visible change in the solution, and the ECM is still a cloudy, homogeneous liquid. If it starts to gel, the viscosity will increase and it will be difficult or impossible to draw the material up into a syringe. If this happens, perform the pH adjustment step on ice, with pre-chilled solutions. Once the injections have been prepared, keep them on ice until use. If the injection is successful, the region around the needle tip whitens and a small bolus is visible. If this is not observed, the injection may have gone into the chamber instead of into the wall. When suturing the animal up after injection, careful closing of the diaphragm is the most important step. If done correctly, the diaphragm will be tight against the lungs and will appear concave. If there is any air left inside the lungs, the diaphragm will remain convex, or have an area where it balloons out. When examining the histology of the injection region at an early time point (30 minutes to 4 hours post-injection), one should see a pink, fibrous area that is devoid of cells this is the reassembled matrix material post-injection (Figure 2). The network often spreads interstitially and may be present throughout many sections. A possible explanation for seeing only a very small area of matrix gel is that a portion of the injection missed the intramural target and was either injected into the LV chamber or leaked out of the injection location. Additionally, depending on the gelation time, the interstitial spread may vary.
Figure 1. Polyacrylamide gel electrophoresis (PAGE) results. (A) Molecular weight standard. (B) Rat tail collagen Type I (2.5 mg/mL) compared to the solubilized human (C) and porcine (D) pericardial ECM (7 mg/mL). Note the presence of collagen as well as several other proteins and peptides in the pericardial matrix samples.
Table 1. ECM components identified with mass spectroscopy.
Figure 2. Myocardial injections: in vivo gelation. H&E stain of human (A) and porcine (B) pericardial matrix injections that have gelled in vivo after 45 minutes. Arrows denote matrix location, stained lighter pink than the myocardium. Scale bar is 500 μm.
Figure 3.Vascular cell infiltration. Fluorescent stains for vessels in the injected human (A-C) and porcine (D-F) matrix gels at two weeks. Endothelial cells are labeled green (A, D), while smooth muscle cells are labeled red (B, E). Merged images are shown in C and F. Scale bar is 100 μm. The white dotted lines indicate the area of matrix injection, as determined by H&E analysis of a nearby section.
Figure 4. Stem cells within matrix injection region. A Hoescht stain for nuclei (blue) and c-kit (green) identifies stem cells in the human (A) and porcine (B) matrix injection regions. Scale bar is 50 μm.
This method allows for the generation of biologically derived, injectable scaffolds for myocardial tissue engineering. Although these methods were initially developed for the fabrication and in vivo testing of a myocardial matrix gel and presented here with a pericardial matrix gel, this protocol can be adapted for use with any tissue, provided the tissue can be appropriately decellularized. Decellularization should be performed and verified prior to the use of these methods, as the presence of DNA in the matrix gel could cause an unwanted immune response. There are a variety of ways to decellularize material, and several good reviews have been written on the subject 3, 4.
When preparing a matrix gel, it is essential that the sample is fully lyophilized, as any remaining moisture will prevent milling. Also, to avoid inhalation of ECM powder, always wear a face mask when milling. Finally, it is important to note that when the matrix gel is intended for an in vivo study, the preparation should be performed in as sterile a manner as possible in order to decrease the risk of contamination that could lead to infection. If modifying this protocol for a different tissue type, certain parameters will need to be optimized. For example, one could change the concentration of ECM some tissues require a higher concentration to form a gel and some may gel at lower concentrations. Additionally, the necessary digestion time and pepsin concentration may vary if the strength of the pepsin used is significantly different.
The small animal surgery model described in this protocol can be utilized for the injection of any in situ gelling material into the LV free wall. To examine cell-based tissue engineering therapies in the heart, this protocol can used to inject a combination of cells and these biologically derived gels. Thus, these methods provide a flexible framework for the preparation and in vivo characterization of biologically derived gels for myocardial tissue engineering.
The authors have nothing to disclose.
This research was supported in part by the NIH Director’s New Innovator Award Program, part of the NIH Roadmap for Medical Research, through grant number 1-DP2-OD004309-01. S.B.S-N. would like to thank the NSF for a Graduate Research Fellowship.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Reagents: | ||||
Pepsin | Sigma-Aldrich | p6887-1G | Lyophilized | |
Biotin | Thermo Scientific | 21217 | ||
Neutravidin-HRP | Thomas Scientific | 21130 | ||
Equipment: | ||||
Wiley Mini Mill | Thomas Scientific | 3383L10 | ||
Labconco Lyophilizer | Labconco, Inc | 7670520 | ||
Surgical supplies: | ||||
Betadine | Purdue Products, L.P. | 67618-154-16 | ||
Lactated Ringers Solution | MWI | 003966 | ||
KY Jelly | MWI | 28658 | ||
Lidocaine, 2% | MWI | 17767 | ||
Buprenorphine hydrochloride | Reckitt Benckiser Healthcare (UK) Ltd. | 12496-0757-1 | ||
Artificial tear ointment | Fisher | NC9860843 | ||
Triple antibiotic ointment | Fisher | 19082795 | ||
Isoflurane | MWI | 60307-120-25 | ||
Otoscope | MWI | 008699 | ||
Stop cock | MWI | 006245 | ||
3-0 Vicrile suture | MWI | J327H | ||
5-0 Proline suture | MWI | s-1173 | ||
Reverse cutting (RC) needle | Ethicon | 8684G | ||
Microhemostats | Fine Science Tools | 13013-14 | ||
Rat tooth microforceps | Fine Science Tools | 11084-07 | ||
No. 10 scalpel | Fine Science Tools | 10110-01 | ||
Blunt scissors | Fine Science Tools | 14108-09 | ||
Sharp, curved scissors | Fine Science Tools | 14085-08 | ||
Large, serrated forceps | Fine Science Tools | 1106-12 | ||
PE160 suction tubing | BD | 427430 | ||
Clippers | MWI | 21608 | ||
Skin staples/stapler | Ethicon | PRR35 | ||
General supplies: | ||||
Stir plates | ||||
0.1 M HCl | ||||
1 M NaOH | ||||
10x PBS | ||||
1x PBS | ||||
70% Ethanol | ||||
0.1 mL syringes | ||||
10 mL syringe | ||||
Q-tips | ||||
Surgical glue | ||||
Surgical drape | ||||
Towel clamps | ||||
Small hand-held vacuum |