The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

Summary

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

Abstract

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

Introduction

Over the past few years, regenerative plastic surgery has emerged as an additional pillar of plastic surgery¹. Regenerative plastic surgery aims to restore damaged tissue by transferring soluble factors, cells, and tissue harvested from the patient to promote tissue restoration in a minimally invasive manner². Adipose-derived stem cells (ASCs) have gained attention due to their ability to differentiate into multiple mesenchymal lineages, making them a promising candidate for regenerative medicine research³. Their cytokine profile displays angiogenic, immunosuppressive, and antioxidative effects⁴.

Traditionally, ASCs were isolated from adipose tissue using an enzymatic approach with collagenase, resulting in a stromal vascular fraction (SVF), which was subsequently cultured to obtain ASCs. These laboratory-based technologies are costly, time-consuming, and importantly, subject to strict regulatory restrictions, complicating clinical translation^5,6,7. In contrast, mechanically isolated stromal vascular fraction (mSVF) protocols offer the clinical benefits of not only bypassing regulatory issues but also minimizing contamination risks^8,9.

Numerous protocols to mechanically isolate the SVF have been described¹⁰. Amongst these, the shifting protocol published by Tonnard et al. has gained the most attention amongst regenerative surgeons¹¹. The fat collected through standard liposuction procedures, known as lipoaspirates, can be transferred between two handheld syringes attached to a connecting device, resulting in a liquid form referred to as nanofat. The obvious benefits of these mSVF isolation protocols include reduced processing time, minimal risk of contamination, as the whole procedure is done in a well-controlled environment, and possible immediate clinical translation¹².

Preclinical and clinical evidence indicates that the properties of mSVF, including cell viability and wound healing properties, are comparable to standard enzymatic isolation methods¹². The potential of mSVF in promoting wound healing in rat and murine models was validated through in vivo studies by Chen et al. and Sun et al.^13,14. However, there is a lack of available data regarding wound healing in the clinical setting. Promising results were reported when a study group performed autologous fat transplantation in an 83-year-old patient who had a wound with an exposed implant in an open fracture of the lower extremity¹⁵. Furthermore, Lu et al. conducted a comparison between mSVF and negative pressure wound therapy in a cohort of 20 patients with chronic wounds¹⁶. Their findings revealed that mSVF treatment resulted in a higher rate of wound healing compared to negative pressure wound therapy¹⁶. Both mentioned studies injected mSVF alone or in combination with a gel into the targeted wound area^15,16.

In the real-world scenario, clinical application of mSVF is limited due to unpredictable absorption rates at recipient sites^17,18. Scaffolds promise a remedy to this issue, as they assist in cell retainment, vascularization, and integration into the surrounding tissue^19,20,21. Fibrin hydrogels are a commonly used, FDA-approved tool used in surgical disciplines and have been shown to be an effective carrier of mSVF¹⁹. Fibrin gel is a biopolymeric material which provides several advantages in functioning as a cell carrier: it displays excellent biocompatibility, promotes cell attachment, and is capable of degrading in a controllable manner^22,24,25. Additionally, it demonstrates minimal inflammatory and foreign body reaction and is easily absorbed during the natural course of wound healing²². We believe that the diverse regenerative capabilities of mSVF cells mentioned and the advantageous combination with a fibrin hydrogel can provide an innovative approach to enhance wound healing processes. Overall, this approach allows for an efficient topical delivery of viable mSVF cells. We hereby present the protocol that combines mSVF with a fibrin hydrogel intended for application in wound healing purposes.

Protocol

This study was performed in accordance with the Declaration of Helsinki. All adult donors provided written informed consent to allow further use of the collected tissue samples. The protocol follows the guidelines of our institution's human research ethics committee.

1. Harvest of adipose tissue

1. Harvest the adipose tissue by performing a standard liposuction in a conventional fat-harvesting technique described in previous publications^26,27. Ensure to use a tumescent solution that consists of regular ringer's lactate and epinephrine in a ratio of 1: 200,000.
2. Perform the liposuction with a 4 mm aspiration cannula, under negative pressure and vibration, into a sterile bag. Transfer the harvested fat to the laboratory immediately.

2. mSVF-Isolation

1. Perform the following steps (sections 2 and 3) in a cell culture hood to provide an aseptic work area. Wear a regular lab coat and gloves to ensure biological safety level 2.
2. Prepare the culture medium: Supplement 500 mL of high-glucose Dulbecco's Modified Eagles's Medium (DMEM) with 50 mL of fetal bovine serum (FBS), 5 mL of penicillin-streptomycin.
3. Transfer the lipoaspirate into a 50 mL centrifuge tube.
4. Transfer the lipoaspirate into a sterile 20 mL Luer-lock syringe and attach a 1.4 mm connector. Ensure that no air is inside the syringe.
5. Attach a second 20 mL Luer-lock syringe to the contralateral side of the 1.4 mm connector.
6. Push the adipose tissue from one syringe to the other for a total of 30 times.
7. Transfer the emulsified fat into a fresh 50 mL centrifuge tube.
8. Centrifugate the emulsified fat at 500 x g for 10 min.
9. After centrifugation, discard the oily top layer. Then, collect the central purified mSV-layer. Transfer it into a fresh 50 mL centrifuge tube and discard the aqueous phase.
10. Fill the centrifuge tube with culture medium (from step 2.2) up to the 40 mL mark.
11. Place the centrifuge tube into the centrifuge and once again centrifuge at 500 x g for 5 min.
12. Collect the resulting mSVF-layer and transfer it into a new 50 mL centrifuge tube.

3. Manufacturing of mSVF-fibrin hydrogel

1. Combine 100 µL of mSVF with 10 µL of thrombin (100 U/mL), 10 µL of CaCl₂ (80 mM), and 70 µL of tranexamic acid (100 mg/mL) in a sterile 1.5 mL tube.
2. Use a fresh pipette tip to add 10 µL of fibrinogen (100 mg/mL) as the last component, shortly before application. Beware that hydrogel-polymerization is observed within approximately 10-30 s.
3. For clinical application, perform this last step shortly before topical administration, preferably at the bedside. For analytical purposes, transfer into a 12-well plate by pipetting.

4. Viability assay and histology

1. Pipette 200 µL of the mSVF-hydrogel mixture from step 3.3 into one well of a 12-well plate.
2. Pipette 100 µL of the mSVF-collection (step 2.12.) into one well as a positive control.
3. Pipette 200 µL of fibrin hydrogel only into one well as a negative control.
4. Place the 12-well plate into a regular incubator at 37 °C and 5% CO₂ for 30 min.
5. After this time, add 1 mL of resazurin (alamar blue, 10% concentration, diluted in culture medium) to each well.
6. After the addition, incubate the 12 well-plate at 37 °C and 5% CO₂ for 24 h.
7. After 24 h, measure the first fluorescence intensity using a cell imaging multimode microplate reader using an excitation wavelength of 555 nm and an emission wavelength of 596 nm.
8. Perform consecutive fluorescence intensity measurements on days 3 and 7.
9. If histologic assessment is necessary, stop the experiment on days 1, 3, and 7 and fix the fibrin hydrogel in 4% paraformaldehyde at 4 °C for 24 h. After fixation, add 1% phosphate-buffered saline (PBS) and store it at 4 °C.
10. Embed the hydrogels in an optimal cutting temperature (OCT) compound and section frozen blocks at 10 µm thickness with a cryostat. Stain the sections with hematoxylin and eosin (H&E) solution according to standard protocols²⁸.

Results

Resazurin assay
We first examined the in vitro cell viability of the mSVF cells. For this purpose, we conducted a resazurin cell viability assay on days 0, 3, and 7. The cell viability at days 0, 3, and 7 of a total of four samples are shown in Figure 1. The values of day 0 serve as the baseline and were set as 100%. At day 3, the positive control (mSVF) showed a slight decrease to 78.92% (± 5.33%), while the mSVF-fibrin hydrogel combination remained at 9...

Discussion

The mechanical isolation of SVF provides an elegant alternative to the traditional enzymatic approach and offers broad access for clinical application²⁹. In fact, mSVF, as proposed in the present manuscript, is already in clinical use for soft tissue treatment of scars or as an adjunct for cosmetic procedures³⁰. The protocol presented here provides a simple method for efficient topical delivery of viable mSVF cells. While the positive control with only mSVF cells showed a t...

Materials

Name	Company	Catalog Number	Comments
12-Wellplate	Sarstedt	83.3921
4′,6-diamidino-2-phenylindole (DAPI)	Biochemica	A1001.0010
50 mL-Falcon	Falcon	352070
Absorbent Towels, Two Pack	Halyard	89701
Alamar blue 25 mL	Invitrogen	DAL1025
Albumin, Bovine (BSA)	VWR	0332-500G
Biotek Cytation 5	Agilent		Cell Imaging Multimode Microplate Reader
CaCl2	Sigma-Aldrich	C5670-500G
Cryostat	Microtome
DMEM with 4,5 g/L glucose,with L-Glutamine, with sodium pyruvate	VWR	392-0416
DPBS	Gibco	14190-144
Epinephrin	Sigma-Aldrich	E4250
Fetal Bovine Serum	Biowest	S181H-500
Fibrinogen Human Plasma 100 mg	Sigma-Aldrich	341576-100MG
Formalin	Fisher Scientific	SF100-4
Formalin 4%	Formafix	1308069
FSC 22-Einbettmedium, blau	Biosystems	3801481S
Hematoxylin & Eosin Solution	Sigma-Aldrich	H3136 / HT110132
Lactated Ringer’s Solution 1000 mL	B Braun	R5410-01
Mercedes Cannula 4mm	MicroAire	PAL-R404LL
NaCl 0.9%	Bbraun	570160
OCT Embedding Matrix 125 mL	CellPath	KMA-0100-00A
Paraformaldehyde	Fisher Scientific	10342243
PBS 1%	Sigma-Aldrich	P4474
PenStrep	Sigma-Aldrich	P4333-100ML
Petridish 150mm	Sarstedt	83.1803
Phalloidin-iFluor 488 Reagent	Abcam	ab176753
Sterile Syringe 20 mL Luer	HENKE-JECT	5200-000V0
Sterile Syringe 30 mL Luer-Lock	BD	10521
Thrombin from Human Plasma	Sigma-Aldrich	T6884-100UN
Tranexamic acid	Orpha Swiss	6837093
Tulipfilter 1.2	Lencion Surgical	ATLLLL
Tulipfilter 1.4	Lencion Surgical	ATLLLL