Summary
Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.
Abstract
One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.
Introduction
Cardiac stem cell therapy has emerged as a potential approach for myocardial repair and regeneration1,2. Despite the recent positive results in animal models and clinical trials, the application of stem cell-based therapy for myocardial repair is limited due to low retention and poor survival of injected cells at the infarcted heart tissues3,4. As a result, the use of cell-based tissue engineering, including injectable biomaterials5, cardiac patches6, and cell sheets7, has been intensively studied to improve cell retention and integration within the host myocardium.
Among the various potential approaches to bioengineered cardiac tissue repair, injectable hydrogels combined with appropriate cell types, such as mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), are an attractive option to effectively deliver cells into myocardial regions8,9. Gelatin, a well-known natural polymer, can be used as an injectable matrix due to its great biocompatibility, considerable biodegradability, and reduced immunogenicity when compared with a wide range of biomaterials used in biomedical applications. Although gelatin-based injectable platforms have great potential, their applicability in vivo remains limited based on their low mechanical stiffness and easy degradability in the physiological environment.
To overcome these limitations, a novel and simple design of gelatin-based hydrogels consisting of hydroxyphenyl propionic acid has been proposed for in vivo applications. Gelatin-hydroxyphenyl propionic acid (GH) conjugates can be cross-linked in situ in the presence of an enzyme, horseradish peroxidase (HRP), and subsequently encapsulate various drugs, biomolecules, or cells within the hydrogel, suggesting great potential in tissue engineering applications10,11,12,13,14. In addition, we have recently investigated the therapeutic effects of GH hydrogels containing encapsulated MSCs and demonstrated their use in successful cardiac repair and regeneration after MI in a murine model15. In this protocol, we describe a simple technique for the encapsulation and in vitro three-dimensional (3D) proliferation of MSCs within GH hydrogels. We also introduce a surgical procedure designed to generate a murine MI model via coronary artery ligation and intramyocardial transplantation of MSC-loading GH hydrogels into the infarcted heart.
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Protocol
All animal research procedures were provided in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent Experiments provided by the Institutional Animal Care and Use Committee (IACUC) in the School of Medicine of The Catholic University of Korea.
1. Preparation of MSCs and injectable gelatin hydrogels
- Culture MSCs in a 100 mm culture dish at 37 °C and 5% CO2. When MSCs growth reaches 80% confluence, wash the dish twice with DPBS and add 1 mL of trypsin-substitute at 37 °C for 3 min.
NOTE: MSCs were isolated from murine bone marrow following conventional procedures16, cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic−antimycotic solution, and used between passage 7‒9 for this study. - Add 9 mL of culture medium and centrifuge at 500 x g for 3 min. Next, discard the resulting supernatant, resuspend the cells in 1 mL of PBS, and maintain the cell suspension on ice.
- Dilute 10 µL of cell suspension with 10 µL of Trypan blue and obtain the cell concentration using an automated cell counter.
- Resuspend and transfer MSCs to a 1 mL tube at a density of 1 x 107 cells/mL.
- Prepare a 6.25 wt% of GH conjugate solution in PBS and separate into 2 vials. Next, mix the GH solutions with either 6 µg/mL of HRP (GH solution A) or 0.07 wt% of H2O2 (GH solution B).
NOTE: Prepare gelatin-hydroxyphenyl propionic acid (GH) conjugates according to published protocols12,15.- Keep a 9:1 volumetric ratio of GH conjugate solution to HRP (GH solution A) and GH conjugate solution to H2O2 (GH solution B), respectively.
- Prior to mixing the MSCs with GH solution A, briefly centrifuge the cell suspension at 1,000 x g and carefully aspirate the resulting supernatant. Subsequently, mix the pellet containing MSCs with GH solution A.
2. In situ MSC-loading and three-dimensional in vitro culture
- Load GH solution A (containing MSCs) and GH solution B into either side of a dual syringe. Plate 300 µL of the combined GH solutions with MSCs at a final density of 5 x 106 cells/mL onto an eight-well chamber slide.
- After in situ hydrogel formation and subsequent MSC encapsulation via enzymatic cross-linking, add 700 µL of DMEM containing 10% FBS and 1% antibiotic−antimycotic solution.
- Incubate the slide at 37 °C and 5% CO2 and replace the culture medium every 2‒3 days.
3. Confirmation of in vitro proliferation and survival of MSCs within GH hydrogels
- To determine the viability of 3D cultured MSCs within GH hydrogels, use a live/dead cell staining assay after the predetermined incubation time.
- Following incubation of the encapsulated MSCs in GH hydrogels for 3, 5, 7 or 14 days, aspirate the medium and wash the well twice with PBS.
- Prepare a staining solution containing 5 µL of calcein AM and 20 µL of ethidium homodimer-1 (EthD-1) in 10 mL of DPBS.
- Add 200 µL of the staining solution to the well and incubate for 30 min in the dark at room temperature.
- Aspirate the staining solution and wash the well twice with PBS.
- Carefully separate the chamber from the slide and place a full coverslip over the GH hydrogels. Use a confocal microscopy to visualize the degree of proliferation and morphological changes of the encapsulated MSCs.
NOTE: Fluorescent images were acquired under 200x magnification and imaged at the excitation/emission wavelengths of 470/540 nm for calcein and 516/607 nm for EthD-1.
4. Induction of myocardial infarction in mice
- Anesthetize 7-week-old male C57BL/6 mice (20‒22 g) with intraperitoneal injection of a mixture of Zoletil (30 mg/kg) and Rompun (10 mg/kg) in saline.
- Prior to surgery, depilate the mouse chest using hair removal cream and sterilize the skin with iodine.
- Place the mouse on an operating table and intubate by inserting a catheter into the trachea to provide supplemental oxygen via mechanical ventilation.
- Gently cut through the skin using surgical scissors and then penetrate the intercostal muscles by micro scissors. Separate the 2nd and 3rd left ribs using a 5-0 silk suture to maintain an open chest cavity.
- Carefully ligate the left anterior descending (LAD) coronary artery using a needle holder with an 8-0 polypropylene suture and cut the suture using electrocautery.
- Observe an immediate color change in the anterior left ventricular wall.
5. Intramyocardial transplantation of MSC-loading GH hydrogels
- After inducing the myocardial infarction by LAD ligation, inject 10 µL of MSC-loading GH solutions into two different points at the infarct border zone (total: 2 x 105 MSCs/20 µL) using a dual-syringe equipped with a 26G needle.
- Following the same procedure described in Step 1, prepare and transfer MSC-loading GH solutions into a dual syringe.
NOTE: To assess the engraftment of MSC-loading GH hydrogels within the infarcted area, MSCs and GH conjugates were pre-labeled with PHK26 and fluorescein isothiocyanate (FITC), respectively.
- Following the same procedure described in Step 1, prepare and transfer MSC-loading GH solutions into a dual syringe.
- Restore the opened chest cavity and close the muscles and skin using 5-0 sutures.
NOTE: Prior to chest closure, remove the air using a catheter syringe. - Remove the tracheal tube and place the mouse in a cage under an infrared lamp during recovery.
- For post-operative analgesia, administer subcutaneous Ketoprofen injections (5 mg/kg per day) for a minimum of 72 h. All mice should be closely monitored for an appropriate time to ensure proper recovery after surgical procedures as well as adequate pain treatment.
6. Echocardiography
- Four weeks following transplantation, initially anesthetize the mouse with 5% isoflurane and then adjust the isoflurane concentration to 1%.
- Depilate the chest using hair removal cream and place the mouse on a heating pad. Apply ultrasound transducer gel onto the chest.
- Acquire two-dimensional parasternal short axis views and record M-mode tracings at the level of the papillary muscle.
NOTE: Place a linear array transducer (7‒15 MHz) in the left parasternal line and view the anatomical structures. - Measure corresponding lines for LVAW, LVID, and LVPW to obtain cardiac wall thickness, chamber dimension, and fractional shortening.
NOTE: Compare cardiac function including the ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV) at the level of the papillary muscle to ensure proper assessment at the same anatomic location.
7. Histological evaluation
- At the predetermined time after transplantation of MSC-loading GH hydrogels into the infarcted heart, euthanize the mouse in a CO2 chamber and collect the heart for histological analysis15.
- For hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining, fix the dissected heart tissues in 4% paraformaldehyde (PFA) and embed in paraffin. Next, cut paraffin-embedded heart blocks into 4 µm serial sections using a microtome and stain the sections with MT stain according to standard protocols17.
- Acquire images on a slide scanner at 20x magnification and calculate the infarct size of the treatment groups.
Infarct size (%) = total infarct circumference / total LV circumference x 100 - Calculate both circumferences by midline length measurement. For LV midline circumferences, measure the centerline lengths between the endocardial and epicardial surfaces. For midline infarct circumferences, measure the lengths of infarct including more than 50 % of the whole thickness of myocardium18.
NOTE: All image analyses were performed using ImageJ software. - Measure the wall thickness of the scar at the papillary muscle levels.
- Calculate the fraction of collagen area.
Collagen area (%) = total area of interstitial fibrosis/myocyte area x 100
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Representative Results
To effectively deliver MSCs to the infarcted myocardium, MSC-loading in situ cross-linkable hydrogels described in Figure 1 were used in this protocol. Prior to in vivo transplantation, the proliferation and survival of MSCs in GH hydrogels were confirmed by a 3D in vitro live/dead cell staining assay (live: green; dead: red). As shown in Figure 2, representative images exhibited sufficient MSCs proliferation, showing branched networks within GH hydrogels. In addition, an extensive multicellular 3D structure of MSCs was clearly observed at day 14, indicating that GH hydrogels could provide a proper microenvironment for the encapsulated cells.
After the induction of MI via LAD ligation, MSC-loading GH hydrogels were intramyocardially transplanted into peri-infarct areas (Figure 3A). As shown in Figure 3B, the MSCs and gel were appropriately sustained within the infarcted region. MSCs, stained with PHK26 (red), were well integrated into GH hydrogels, stained with FITC (green), presenting successful engraftment and retention in the infarcted hearts for in vivo application.
To verify the therapeutic effects of MSC-loading GH hydrogels in a murine MI model, the changes in cardiac function and structure were evaluated by echocardiography and histological analysis at day 28 post-transplantation and compared among the different treatment groups. The representative echocardiography showed improved cardiac functions, including FS, EF, and ESV, in the MSC/gel treated group compared with the other groups (Figure 4). In addition, histological analysis exhibited less fibrosis, thicker infarcted walls, and a smaller infarct size in the MSC/gel treated group than in the other groups, indicating that this protocol contributed beneficial effects by significantly attenuating LV remodeling (Figure 5).
Figure 1: Scheme of the process for improving stem cell retention and engraftment using injectable hydrogels. In situ cross-linkable GH hydrogels containing bone marrow-derived MSCs were prepared and transplanted by intramyocardial injection into the infarcted heart. Please click here to view a larger version of this figure.
Figure 2: In vitro 3D MSC proliferation within GH hydrogels. Representative images of live (green)/dead (red) MSCs obtained via a confocal microscopy following live/dead cell staining after 3, 5, 7, and 14 days of incubation (200x magnification; scale = 100 µm). The images and video were partly adapted with permission from Kim et al.15. Please click here to view a larger version of this figure.
Figure 3: In vivo transplantation of MSC/Hydrogels. (A) A schematic diagram showing intramyocardial transplantation after the induction of MI. (B) Representative images of transplanted MSCs and GH hydrogels labeled with PKH26 (red) and FITC (green), respectively. Mice were sacrificed after 1, 3, 5, or 7 days of transplantation and their hearts were then excised to assess the degree of MSC and GH hydrogel engraftment. The excised hearts were cryo-fixed, prepared into serial-sections, and imaged via a confocal microcopy (200x magnification; scale = 100 µm). The images were partly adapted with permission from Kim et al.15. Please click here to view a larger version of this figure.
Figure 4: Improvements in cardiac function following MSC/Hydrogels transplantation. (A) Representative video of echocardiography. (B) Representative short axis M-mode image with measurements, including left ventricular anterior wall thickness in diastole (LVAWd) and systole (LVAWs), internal diameter in diastole (LVIDd) and systole (LVIDs), and posterior wall thickness in diastole (LVPWd) and systole (LVPWs). (C‒E) Functional improvements in the ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV) after 28 days of transplantation of all treatment groups. Data were represented as mean ± standard deviation (*p < 0.05, **p < 0.001, ***p < 0.0001; n = 9‒12 per group). The videos and results were partly adapted with permission from Kim et al.15. Please click here to view a larger version of this figure.
Figure 5: Improvements in cardiac structure following MSC/Hydrogels transplantation. (A) Representative images of histological evaluation. (B‒D) Structural improvements were observed in infarct size, along with less infarcted wall thinning and fibrosis. Scale = 1 mm. Data are represented as the mean ± standard deviation (*p < 0.05, **p < 0.001, ***p < 0.0001; n = 4‒7 per group). The images and results were partly adapted with permission from Kim et al.15. Please click here to view a larger version of this figure.
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Discussion
Injectable GH hydrogels have great potential for in vivo applications because of their ability to homogenously incorporate diverse therapeutic agents in situ. Furthermore, their physical and biochemical properties can be easily manipulated based on disease-dependent requirements. In this respect, injectable hydrogels have been proposed to address the major limitations in current cardiac stem cell therapy hampered by poor survival and cell retention (i.e., < 10% within 24 h post-transplantation) in the injured heart19,20. To overcome this poor outcome, the protocol described herein provides a simple and feasible method to improve cell retention and survival using GH hydrogels that can be cross-linked in situ after myocardial transplantation, which have demonstrated favorable effects on cardiac structure and function in a murine MI model.
The primary advantage feature of this technique is its broad in vivo applicability with any type of cell and biomolecule, which can be loaded by simply mixing with the pregel GH solution prior to injection. Furthermore, to obtain a comprehensive understanding of donor-to-host interactions, a straightforward labeling approach of the GH conjugates and/or encapsulated biomolecules can be adapted to track changes in their in vivo stability, host integration, and resorption kinetics. To our knowledge, the use of injectable gelatin-based hydrogels combined with therapeutic stem cells was the first to validate the restorative potential of cardiac tissue in vitro and in vivo15.
At the current stage in this research, GH hydrogels that are loaded with MSCs, injected, and cross-linked in situ were used as a proof of concept to assess their applicability in a murine MI model. Although this method seemingly improved MSC engraftment and retention in the transplanted heart tissues, the detailed conditions during injection should be considered for optimizing therapeutic efficacy, such as the location of injection site (i.e., peri-infarct zone or infarct zone), volume and number of injections, and stiffness of the hydrogel (i.e., hard to inject or easy to leak).
In conclusion, we have demonstrated a protocol for a representative murine MI model by LAD ligation and a practical method for intramyocardial transplantation of stem cells using in situ cross-linked hydrogels to improve the retention and engraftment of transplanted MSCs. These techniques provide an effective method for intramyocardial transplantation of MSC-loading injectable hydrogels and highlight their great potential for application in large animals and clinical translation.
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Disclosures
The authors have no conflicts of interest to declare with this work.
Acknowledgments
This research is supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Education (NRF-2018R1D1A1A02049346)
Materials
| Name | Company | Catalog Number | Comments |
| 4 % paraformaldehyde (PFA) | Intron | IBS-BP031-2 | |
| 5-0 silk suture | AILEE | SK534 | |
| 8-0 polypropylene suture | ETHICON | M8732H | |
| 8-well chamber slide | Nunc LAB-TEK | 154534 | |
| Angiocath Plus (22GA) catheter | BD Angiocath Plus | REF382423 | |
| Antibiotic-antimyocotic | Gibco | 15240-062 | |
| Centrifuge | GYROGEN | 1582MGR | |
| Confocal microscope | Zeiss | LSM 510 | |
| Cover slipe | MARIENFELD | 101242 | |
| Deluxe High Temperature Cautery kit | Bovie | QTY1 | |
| DMEM | Gibco | 11995-065 | |
| DPBS | Gibco | 14040-133 | |
| Dual-syringe | |||
| EOSIN | SIGMA-ALDRICH | HT110116 | |
| Ethanol | EMSURE | K49350783 739 | |
| FBS | Gibco | 16000-044 | |
| Fechtner conjunctiva forceps titanium | WORLD PRECISISON INSTRUMENTS | WP1820 | |
| Fluorescein isothiocyanate isomer I (FITC) | SIGMA-ALDRICH | F7250 | |
| Forcep | HEBU | HB0458 | |
| Hair removal cream | Ildong Pharmaceutical | ||
| Heating pad | Stoelting | 50300 | Homeothermic Blanket System |
| 50301 | Replacement Heating Pad for 50300 (10 X 12.5cm) | ||
| Hematoxylin | SIGMA-ALDRICH | HHS80 | |
| Horseradish peroxide (HRP; 250-330 U/mg) | SIGMA-ALDRICH | P8375 | |
| Hydrogen peroxide (H2O2; 30 wt % in H2O) | SIGMA-ALDRICH | 216763 | |
| Iodine | Green Pharmaceutical | ||
| LIVE/DEAD cell staining kit | Thermo Fisher | R37601 | |
| Mechanical ventilator | Harvard Apparatus | ||
| Micro centrifuge | HANIL | Micro 12 | |
| Micro needle holder | KASCO | 37-1452 | |
| Micro scissor | HEBU | HB7381 | |
| Microscope | OLYMPUS | SZ61 | |
| MT staining kit | SIGMA-ALDRICH | HT1079-1SET | Weigert’s iron hematoxylin solution |
| HT15-1KT | Trichrome Stain (Masson) Kit | ||
| Paraffin | LK LABKOREA | H06-660-107 | |
| PBS buffer | Gibco | 10010-023 | |
| PHK26 staining kit | SIGMA-ALDRICH | MINI26 | |
| Slide scanner | Leica | SCN400 | |
| Surgical scissor | HEBU | HB7454 | |
| Surgical tape | 3M micopore | 1530-1 | |
| Tissue cassette | Scilab Korea | Cas3003 | |
| Transducer gel | SUNGHEUNG | SH102 | |
| Trout-Barraquer needle holder curved | KASCO | 50-3710c | |
| Ultrasound system | Philips | Affiniti 50 | |
| Xylene | JUNSEI | 25175-0430 |
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