Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

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 regeneration^1,2. 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 tissues^3,4. 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 regions^8,9. 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 applications^{10,11,12,13,14}. 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.

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

1. 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.
2. 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.
3. Dilute 10 µL of cell suspension with 10 µL of Trypan blue and obtain the cell concentration using an automated cell counter.
4. Resuspend and transfer MSCs to a 1 mL tube at a density of 1 x 10⁷ cells/mL.
5. 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 protocols^12,15.
   1. 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.
6. 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

1. 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.
2. 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.
3. 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

1. To determine the viability of 3D cultured MSCs within GH hydrogels, use a live/dead cell staining assay after the predetermined incubation time.
2. 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.
3. Prepare a staining solution containing 5 µL of calcein AM and 20 µL of ethidium homodimer-1 (EthD-1) in 10 mL of DPBS.
4. Add 200 µL of the staining solution to the well and incubate for 30 min in the dark at room temperature.
5. Aspirate the staining solution and wash the well twice with PBS.
6. 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

1. 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.
2. Prior to surgery, depilate the mouse chest using hair removal cream and sterilize the skin with iodine.
3. Place the mouse on an operating table and intubate by inserting a catheter into the trachea to provide supplemental oxygen via mechanical ventilation.
4. 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.
5. 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.
6. Observe an immediate color change in the anterior left ventricular wall.

5. Intramyocardial transplantation of MSC-loading GH hydrogels

1. 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.
   1. 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.
2. 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.
3. Remove the tracheal tube and place the mouse in a cage under an infrared lamp during recovery.
4. 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

1. Four weeks following transplantation, initially anesthetize the mouse with 5% isoflurane and then adjust the isoflurane concentration to 1%.
2. Depilate the chest using hair removal cream and place the mouse on a heating pad. Apply ultrasound transducer gel onto the chest.
3. 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.
4. 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

1. 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.
2. 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.
3. 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
4. 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.
5. Measure the wall thickness of the scar at the papillary muscle levels.
6. Calculate the fraction of collagen area.
   Collagen area (%) = total area of interstitial fibrosis/myocyte area x 100

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 ad...

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 heart

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