Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Summary

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

Abstract

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

Introduction

Tissue engineering focuses on the fabrication of tissue and organ substitutes that maintain, restore or augment the function of inoperable in vivo counterparts^1,2. The fate of engineered tissue constructs crucially depends on an adequate vascularization³. Microvascular networks within these constructs should be hierarchically organized with arterioles, capillaries, and venules to allow efficient blood perfusion after inosculation to the recipient's vasculature⁴. The generation of such networks is among the key challenges in tissue engineering. For this purpose, a broad spectrum of experimental vascularization strategies has been introduced over the last two decades^5,6.

Angiogenic approaches stimulate the ingrowth of recipient microvessels into engineered tissues by means of structural or physicochemical scaffold modification, such as the incorporation of growth factors⁷. However, for the vascularization of large three-dimensional constructs, angiogenesis-dependent strategies are markedly limited by slow growth rates of developing microvessels⁸.

In contrast, the concept of prevascularization aims for the generation of functional microvascular networks within tissue constructs prior to their implantation⁹. Conventional prevascularization involves the co-culture of vessel-forming cells, such as endothelial cells, mural cells or stem cells¹⁰, within scaffolds. After microvascular network formation, the prevascularized constructs can then be implanted into tissue defects. Noteworthy, this prevascularization approach is difficult to apply in a clinical setting, because it is based on complex and time-consuming in vitro procedures, which are restricted by major regulatory hurdles⁹. Accordingly, there is still a need for the development of novel prevascularization strategies that are more suitable for a broad clinical application.

Such a prevascularization strategy may be the application of adipose tissue-derived microvascular fragments (ad-MVF). ad-MVF represent potent vascularization units that can be harvested in large amounts from the fat tissue of rats^11,12 and mice¹³. They consist of arteriolar, capillary, and venular vessel segments, which exhibit a physiological microvessel morphology with a lumen and stabilizing perivascular cells^14,15. This unique feature allows the immediate implantation of ad-MVF-seeded scaffolds into tissue defects without precultivation. There, the ad-MVF rapidly reassemble into functional microvascular networks. Furthermore, ad-MVF represent a rich source of mesenchymal stem cells¹⁶, which may additionally contribute to their striking regenerative capacity. Accordingly, ad-MVF are increasingly used in different fields of tissue engineering^{14,15,17,18,19,20,21}.

The isolation of ad-MVF has originally been established in rats^11,12. Herein, we describe a protocol, which allows the standardized isolation of murine ad-MVF from epididymal fat pads. This may provide further insights into molecular mechanisms underlying ad-MVF function by using transgenic mouse models.

Protocol

All procedures were performed according to the National Institute of Health guidelines for the use of experimental animals and followed institutional guidelines (Landesamt für Soziales, Gesundheit und Verbraucherschutz, Abt. Lebensmittel- und Veterinärwesen, Zentralstelle, Saarbrücken, Germany).

1. Preparation of Surgical Instruments

1. Keep ready the dissection scissors, surgical forceps, small preparation scissors, fine forceps and a sterile Petri dish with 15 mL Dulbecco's modified Eagle medium (DMEM; 10% fetal calf serum (FCS), 100 U/mL penicillin, 0.1 mg/mL streptomycin) for harvesting epididymal fat pads.
2. Expose the surgical instruments to a disinfecting solution for 5 min. Alternatively, sterilize them (steam sterilization; 121 °C, 20 min).

2. Animals and Anesthesia

1. Choose carefully the strain of the mice as indicated for the study and the subject under investigation.
   NOTE: In this study we used male wild-type C57BL/6 mice as donors for the harvesting of epididymal fat. Of interest, ad-MVF may be also isolated from transgenic green fluorescent protein (GFP)-positive donor animals (C57BL/6-Tg(CAG-EGFP)1Osb/J)²². This bears the major advantage that the fragments are easily detectable by immunohistochemical staining of GFP after implantation into GFP-negative wild-type mice¹⁴.
2. Anesthetize the animals with an intraperitoneal injection of xylazine (15 mg/kg) and ketamine (75 mg/kg). Make sure that the animals are deeply anesthetized by performing a toe pinch with no response. Eye lubricant is not indicated as the donor animals are sacrificed after fat harvesting.
   NOTE: Ensure that analgesia and surgical sterility are in agreement with the respective guidelines of the country and institution where the experiments are planned.

3. Harvesting of Epididymal Fat Pads

1. Transfer the animal to an operation table. Place the animal in a supine position under a surgical stereomicroscope and confirm deep anesthesia using toe pinch.
2. Immobilize the paws by taping them to a surgical drape and disinfect the abdomen with disinfecting solution.
3. Separate the abdominal skin free of the underlying muscle layer with the dissection scissors.
4. Perform a midline laparotomy with the dissection scissors and laterally unfold the flaps of the abdominal wall.
5. Bilaterally identify testis, epididymis and the epididymal fat pad using the fine forceps (Figure 1). Do not harm the intestinal structures to prevent fecal contamination of the fat pads.
6. Harvest the epididymal fat pads with the small preparation scissors and the fine forceps under the stereomicroscope. Keep a safety margin of several mm between the epididymis and the fat to reduce the risk of accidental epididymal harvesting.
   NOTE: This procedure can also be performed without a stereomicroscope. However, this may further increase the risk of accidental injury of the epididymis and spermatic cords.
7. Transfer the epididymal fat pads into a Petri dish containing 15 mL of DMEM pre-heated at 37 °C for the transport to the cell laboratory.
8. Sacrifice the animal by incision of the abdominal aorta or cervical dislocation.

4. Isolation of ad-MVF

1. Prepare three sterile Petri dishes with 15 mL of phosphate-buffered saline (PBS), sterile 14-mL polypropylene (PP) tubes, a sterile 50-mL Erlenmeyer flask, sterile 1.5-mL conical microcentrifuge tubes and sterilized fine scissors.
2. Wash the fat pads thrice in Petri dishes with 15 mL of PBS under a laminar flow hood.
3. Transfer the fat into a 14-mL PP tube. Determine the volume of harvested fat tissue (in mL) by means of the tube scale. Mince the fat tissue mechanically with the fine scissors until a homogeneous tissue suspension is obtained.
4. Transfer the minced tissue with two volumes of collagenase NB4G (0.5 U/mL PBS) into a 50-mL Erlenmeyer flask by means of 10-mL measuring pipette. The total volume becomes thrice the volume of fat tissue measured in step 4.3. Perform tissue digestion in an incubator for 10 min under vigorous stirring by means of an automated stirrer (size of magnetic stir bar: 25 mm) at 37 °C and humidified atmospheric conditions with 5% CO₂.
5. Observe a small fraction (10 µL) of the digested tissue under a microscope to judge whether the digestion can be stopped. Ascertain that the digestate mainly contains "free" ad-MVF next to single cells, indicating the appropriate point to stop the enzymatic digestion process (Figure 2).
   NOTE: This step requires experience with the procedure and is critical for the quality of isolated ad-MVF. Prolonged fat digestion results in a single-cell suspension without ad-MVF.
6. Neutralize the enzyme with two volumes of PBS/20% FCS. The total volume becomes thrice the volume of the cell-vessel suspension in step 4.4. Transfer the cell-vessel suspension back into new PP tubes.
7. Incubate the suspension for 5 min at 37 °C to separate ad-MVF from remaining fat by gravity. Then carefully remove the main fat supernatant with a 1-mL precision pipette. Repeat this cycle several times with a 100-µL precision pipette until the suspension appears to be fat-free.
8. Put a 500-µm filter on top of a 50-mL conical centrifuge tube. Transfer the cell-vessel suspension with a 10-mL measuring pipette from the 14-mL PP tubes onto the filter membrane to remove remaining fat clots. Transfer the filtered suspension into new 14-mL PP tubes according to the number of individual ad-MVF isolates for the planned experiments.
   NOTE: For in vitro assays focusing on microvascular network formation, it may be beneficial to further purify the cell-vessel suspension to improve the imaging quality during microscopic analyses. For this purpose, the suspension may be additionally filtered once with a 20-µm filter to remove single cells from the ad-MVF collected on the filter.
9. Centrifuge the cell-vessel suspension (600 x g, 5 min, room temperature) to obtain a pellet containing ad-MVF.
10. After centrifugation, remove the supernatant until 1 mL is left. Resuspend the pellet with ad-MVF in this 1 mL and transfer the suspension into a 1.5-mL conical microcentrifuge tube.
11. Centrifuge the ad-MVF suspension in the microcentrifuge tube (600 x g, 5 min) to obtain a pellet.
12. Remove the supernatant to resuspend the pellet in the required final volume of PBS / 20% FCS.
    NOTE: The number of ad-MVF per isolate can be assessed by microscopic counting. For this purpose, 1/10 of the final cell-vessel suspension is diluted 1:10 in PBS and 100 µL of this dilution are transferred into a well of a 96-well plate. The entire number of ad-MVF exhibiting a vessel-like morphology is then counted and extrapolated to the whole isolate. The required ad-MVF concentration and purification can be individually adapted to the respective in vitro or in vivo application of the ad-MVF. This can include the embedding of ad-MVF in collagen gels for the in vitro analysis of microvascular network formation or the seeding of ad-MVF on scaffolds for in vivo implantation into tissue defects.

Results

In the present study we performed six ad-MVF isolation procedures with fat tissue from 7- to 12-month-old male wild-type C57BL/6 mice (mean body weight: 35 ± 1 g). Figure 1 illustrates the harvesting of murine epididymal fat pads with subsequent mechanical and enzymatic ad-MVF isolation. The time required for the harvesting of fat was 30 min and for the isolation of ad-MVF was 120 min. In total, the procedure took 150 min.

Discussion

In this study we present a well-established protocol for the isolation of ad-MVF. Obtaining ad-MVF from murine adipose tissue is a straightforward procedure with a few critical steps. Mice exhibit different subcutaneous and intraabdominal fat deposits. As previously described for rats, the most suitable fat source for the isolation of ad-MVF are the epididymal fat pads due to their size, homogeneous structure and minimal contamination with larger blood vessels^11,12

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5-mL conical microcentrifuge tube	VWR, Kelsterbach, Germany	700-5239
100-µL precision pipette	Eppendorf, Hamburg, Germany	4920000059
10-mL measuring pipette	Costar, Corning Inc., New York, USA	4488
14-mL PP tubes	Greiner bio-one, Frickenhausen, Germany	187261
1-mL precision pipette	Eppendorf, Hamburg, Germany	4920000083
500-µm filter (pluriStrainer 500 µm)	HISS Diagnostics, Freiburg, Germany	43-50500-03
50-mL conical centrifuge tube	Greiner bio-one, Frickenhausen, Germany	227261
50-mL Erlenmeyer flask	VWR, Kelsterbach, Germany	214-0211
96-well plate	Greiner bio-one, Frickenhausen, Germany	65518
cell detachment solution (Accutase)	eBioscience, San Diego, CA USA	00-4555-56
C57BL/6 mice	Charles River, Cologne, Germany	027
C57BL/6-Tg(CAG-EGFP)1Osb/J mice	The Jackson Laboratory, Bar Harbor, USA	003291
CD117-FITC	BD Biosciences, Heidelberg, Germany	553373
CD31-PE	BD Biosciences, Heidelberg, Germany	553354
Collagenase NB4G	Serva Electrophoresis GmbH, Heidelberg, Germany	17465.02	Lot tested by manufacturer for enzymatic activity
Dissection scissors	Braun Aesculap AG &CoKG, Melsungen, Germany	BC 601
DNA-binding dye (Bisbenzimide H33342)	Sigma-Aldrich, Taufkirchen, Germany	B2261
Dulbecco's modified Eagle medium (DMEM)	PAN Biotech, Rickenbach, Germany	P04-03600
Fetal calf serum (FCS)	Biochrom GmbH, Berlin, Germany	S0615
Fine forceps	S&T AG, Neuhausen, Switzerland	FRS-15 RM-8
Fine scissors	World Precision Instrumets, Sarasota, FL, USA	503261
Dermal skin substitute (Integra)	Integra Life Sciences, Sain Priest, France	62021
Ketamine	Serumwerk Bernburg AG, Bernburg, Germany	7005294
M-IgG2akAL488	eBioscience, San Diego, CA USA	53-4724-80
Octeniderm (disinfecting solution)	Schülke & Mayer, Norderstedt, Germany	118211
Penicillin/Streptomycin	Biochrom, Berlin, Germany	A2213
Petri dish	Greiner bio-one, Frickenhausen, Germany	664160
Phosphate-buffered saline (PBS)	Lonza Group, Basel, Switzerland	17-516F
pluriStrainer 20-µm (20 µm filter)	HISS Diagnostics, Freiburg, Germany	43-50020-03
Rat-IgG2akFITC	BD Biosciences, Heidelberg, Germany	553988
Rat-IgG2akPE	BD Biosciences, Heidelberg, Germany	553930
Small preparation scissors	S&T AG, Neuhausen, Switzerland	SDC-15 R-8S
Surgical forceps	Braun Aesculap AG &CoKG, Melsungen, Germany	BD510R
Tape (Heftpflaster Seide) 1.25 cm	Fink & Walter GmbH, Mechweiler, Germany	1671801
Xylazine	Bayer Vital GmbH, Leverkusen, Germany	1320422
α-SMA-AL488	eBioscience, San Diego, CA USA	53-9760-82	Intracellular labeling additionally requires Cytofix/Cytoperm (BD Biosciences, Heidelberg, Germany; #554722)