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Method Article

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

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DOI:

10.3791/55183

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February 9th, 2017

 ,  , 
1Biomedical Engineering, Tulane University

In This Article

Summary

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

Abstract

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

Introduction

Microvascular network growth and remodeling are common denominators for tissue function, wound healing, and multiple pathologies and a key process is angiogenesis, defined as the growth of new blood vessels from existing ones1,2. For tissue engineering new vessels or designing angiogenic based therapies, understanding the importance of the cellular dynamics involved in angiogenesis is critical. However, this process is complex. It can vary at specific locations within a microvascular network and involves multiple cell types (i.e. endothelial cells, smooth muscle cells, pericytes, macrophages, stem cells) and multiple systems (lymphatic networks and neural networks). Although in vitro models have contributed tremendously to examining the relationship between different cells involved in angiogenesis3, their physiological relevance can be undermined due to their limited complexity and the fact that they do not closely reflect an in vivo scenario. To overcome these limitations, three-dimensional culture systems3, ex vivo tissue models4, microfluidic systems5,6, and computational models7 have been developed and introduced in recent years. However, there is still a need for a model with time-lapse capability to investigate angiogenesis in intact microvascular networks ex vivo. The establishment of new time-lapse models for angiogenesis studies with that level of complexity will provide an invaluable tool to understand the underlying mechanisms regulating angiogenesis and to improve therapies.

A potential model that enables the ex vivo investigation of angiogenesis across an intact microvascular network is the rat mesentery culture model8. In recent work, we have demonstrated that blood and lymphatic microvascular networks remain viable after culture. More importantly, the rat mesentery culture model can be used to investigate functional pericyte-endothelial cell interactions, blood and lymphatic endothelial cell connections, and time-lapse imaging. The objective of this paper is to provide our protocol for the time-lapse imaging method. Our representative results document the multiple cell types that remain viable after the stimulation of angiogenesis with serum and offer examples of using this method for quantifying tissue specific angiogenic responses as well as endothelial cell tracking studies.

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Protocol

All animal experiments and procedures were approved by the Tulane University's Institutional Animal Care and Use Committee (IACUC).

1. Surgical Procedure Setup

  1. Autoclave instruments, surgical supplies, and culture supplies prior to surgery. Surgical supplies for each rat include: 1 drape, 1 drape with pre-cut hole (0.5 in x 1.5 in) in the center, gauze pads, and 1 absorbent underpad. Surgical instruments include: 1 scalpel with a number 10 blade, 2 pairs of tweezers, and a pair of fine scissors. Culture supplies include: 1 drape, 1 pair of tweezers, and prepared 6-well plate inserts with polycarbonate filters.
  2. Sterilize a plexiglass platform, a surgical stage and a surgical benchtop space with 70% ethanol. Keep the surgical stage in a sterile bowl until use.
    1. Create a surgical stage by drilling an approximately 2 in by 1 in hole in the center of a 100 mm culture dish. Next, use sandpaper to smooth any sharp edges and add a layer of silicone glue to the hole's edges to create a raised surface for the tissues.
    2. Alternatively, design the surgical stage using CAD software and make by 3-D printing (Figure 1).
  3. Place a sterile absorbent underpad down and lay a plexiglass platform on top of it. Place the drape, without a pre-cut hole, over a heated pad next to the absorbent underpad.
  4. Pre-warm sterile phosphate-buffered saline (PBS), media and saline to 37 °C. Place media and PBS in separate culture dishes atop the heating pad and place saline in a 50 mL conical tube next to the surgical setup.
  5. Make sure all packages are opened prior to the beginning of the surgery to ensure sterile handling of all materials. A complete list of the common tools used in this procedure are listed in the Table of Specific Surgical Materials and Tools.

2. Mesentery Tissue Harvesting

  1. Use adult male Wistar rats (350 ± 25 g; 6 - 8 weeks of age). Other strains and ages of rats can be substituted.
  2. Anesthetize the rat via an intramuscular injection of ketamine (80 mg/kg body weight) and xylazine (8 mg/kg body weight). Confirm the rat is under anesthesia by pinching between the toes to check for a reflex response; there should be none. Pre-emptive analgesia for this terminal procedure is not necessary.
  3. Shave the abdominal region and remove remaining hair using hair removal cream. Wipe abdominal skin twice with 70% isopropyl alcohol followed by povidone-iodine. For the wipes the surgeon should start at the center of the surgical site and move to the outside of the prepared area in a circular manner as to not overlap areas that have been previously scrubbed with the same piece of sterile gauze or sterile cotton swab. Then transfer animal to the sterile surgical setup and place atop the plexiglass platform.
  4. Using a scalpel blade, make a 0.75 in - 1.25 in incision in the gut starting 1 in below the sternum. Be careful not to puncture the bowel or mesentery (1 layer of skin, 1 layer of connective tissue, and 1 layer of muscle).
  5. Place a drape with a pre-cut hole over the incision and place a sterile surgical stage atop the drape. Ensure the opening aligns with the incision. Use sterile cotton-tipped applicators to locate and pull out the ileum through the surgical stage opening.
  6. Pull 6 - 8 mesenteric windows through the stage using cotton-tipped applicators, and be careful not to touch the windows (Figure 1). Tissues are typically harvested from the ileum region of the small intestine starting near the cecum. Keep exposed tissues moist with warmed sterile saline as needed using a sterile syringe to drip the solution.
  7. Euthanize the rat via intracardiac injection of pentobarbital sodium (0.2 mL per rat). Before removing mesenteric windows, ensure the rat is euthanized by palpating the heart; there should be no pulse.
  8. Remove desired mesentery tissues by using tweezers to grab the fat pad and fine scissors to cut the window. Leave a border of fat (2 mm) around the window. Wash tissues once in warmed sterile PBS and once in media.
  9. Return exteriorized ileum to the abdominal cavity and dispose of animal according to institutional guidelines.

3. Mesentery Tissue Culture for Time-Lapse Studies

  1. Transfer autoclaved culture supplies (see section 1.1) and tissues to a sterile laminar flow hood.
  2. Use tweezers to transfer each tissue atop a polycarbonate filter membrane. Grab tissues by the fat pad to avoid damaging the vasculature.
  3. Quickly spread the tissue using the fat pad, being careful not to touch the window. Invert the insert with the tissue into the bottom of a 6-well plate and cover with 3 mL of media (Figure 1). Typical media used for this procedure includes Minimum Essential Media (MEM) with 1% Penicillin Streptomycin (PenStrep) and 10% Fetal Bovine Serum (FBS). Media can be supplemented with other serums and/or growth factors to stimulate angiogenesis.
  4. Repeat steps 3.2 - 3.3 for each tissue and culture in standard incubator conditions (5% CO2, 37 °C) for up to 5 days.

4. Time-Lapse Imaging of Mesentery Tissue

  1. On the day of imaging, supplement the media in each well with conjugated BSI-Lectin and incubate under standard culture conditions for 30 min. Wash tissues twice with lectin-free media. BSI-Lectin stain will remain visible on the mesentery tissue for up to 3 days in culture.
  2. Transfer the plate to a microscope stage. Identify blood and lymphatic vessels based on their morphology and network structure.
  3. Locate a desired network region on each tissue and take images. Take note of the imaging location to ensure the same region will be captured for subsequent images. If using a motorized stage, document the coordinates.
  4. Return tissues to the incubator and continue to culture until desired end point. Repeat steps 4.1 - 4.3 as needed depending on desired experimental time points.

5. Tissue Immunolabeling

  1. BSI-Lectin Labeling
    1. Incubate tissues for 30 min at 37 °C with 1:40 FITC-conjugated lectin in media (2.5 mL antibody solution per well in 6-well plate) followed by two rinses with media. For rinses, add media and then immediately replace.
  2. Live/Dead Labeling
    1. Incubate tissues for 10 min at 37 °C with 1:500 2 mM ethidium homodimer-1 and 1:500 1 mM calcein AM in media (2.5 mL antibody solution per well in 6-well plate) followed by two rinses with media.
  3. BSI-Lectin/NG2 Labeling
    1. Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.
    2. Fix tissues in cold methanol for 30 min at -20 °C. Wash tissues with PBS (3 x 10 min).
    3. For primary antibody labeling incubate tissues for 1 h at room temperature with 1:100 rabbit polyclonal NG2 antibody and 5% normal goat serum (NGS). Wash tissues with PBS (3 x 10 min).
    4. For secondary antibody labeling incubate tissues for 1 h at room temperature with 1:100 goat anti-rabbit CY2-conjugated antibody (GAR-CY2) and 5% NGS. Wash tissues with PBS (3 x 10 min).
    5. Incubate tissues for 30 min at room temperature with 1:40 FITC-conjugated lectin in PBS followed by two rinses with PBS. For rinses, add PBS and then immediately replace.
    6. To mount the slides, cover tissues with 50:50 PBS and glycerol solution and place coverslip on top. Seal the slide edges using nail polish.
  4. LYVE-1/PECAM Labeling
    1. Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.
    2. Fix tissues in cold methanol for 30 min at -20 °C. Wash tissues with PBS + 0.1% saponin (3 x 10 min).
    3. For primary antibody labeling incubate tissues for 1 h at room temperature with 1:200 mouse monoclonal biotinylated CD31 antibody and 1:100 rabbit polyclonal LYVE-1 antibody in PBS + 0.1% saponin + 2% bovine serum albumin (BSA) + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).
    4. For secondary antibody labeling, incubate tissues for 1 h at room temperature with 1:500 CY3-conjugated streptavidin antibody and 1:100 GAR-CY2 in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).
    5. To mount slides, cover tissues with 50:50 PBS and glycerol solution and place a coverslip on top. Seal the slide edges using nail polish.
  5. BrdU/BSI-Lectin Labeling
    1. Add 1 mg/mL BrdU to media and replace tissue media with BrdU solution. Incubate for 2 h at 37 °C.
    2. Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.
    3. Fix tissues in cold methanol for 30 min at -20 °C. Wash tissues with PBS (3 x 10 min).
    4. Denature tissue DNA in 2 M HCl for 1 h at 37 °C. Wash tissues in PBS + 0.1% saponin (3 x 10 min).
    5. For primary antibody labeling, incubate tissues for 1 h at room temperature with 1:100 monoclonal mouse anti-BrdU in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).
    6. For secondary antibody labeling, incubate tissues for 1 h at room temperature with 1:100 goat anti-mouse Cy3-conjugated antibody (GAM-Cy3) in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).
    7. Incubate tissues for 30 min at room temperature with 1:40 FITC-conjugated lectin in PBS followed by two rinses with PBS.
    8. To mount slides, cover tissues with 50:50 PBS and glycerol solution and place coverslip on top. Seal the slide edges using nail polish.
  6. BSI-Lectin/CD11b labeling
    1. Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.
    2. Fix tissues in cold methanol for 30 min at -20 °C. Wash tissues with PBS + 0.1% saponin (3 x 10 min).
    3. For primary antibody labeling incubate tissues for 1 h at room temperature with 1:100 mouse anti-rat CD11b in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).
    4. For secondary antibody labeling incubate tissues for 1 h at room temperature with 1:100 GAM-Cy3 in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).
    5. Incubate tissues for 30 min at room temperature with 1:40 FITC-conjugated lectin in PBS followed by two rinses with PBS.
    6. To mount slides, cover tissues with 50:50 PBS and glycerol solution and place coverslip on top. Seal the slide edges using nail polish.

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Results

After 3 days in culture, tissues were labeled with a live/dead viability/cytotoxicity kit to demonstrate the viability of the microvasculature in the rat mesentery culture model (Figure 2A). The majority of cells present in the mesentery remained viable in the culture where endothelial cells were identified based on their location in microvascular segments. Endothelial cell proliferation was also confirmed by lectin/BrdU labeling (Figure 2D). Smooth muscl...

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Discussion

This protocol documents a method for using the rat mesentery culture model as an ex vivo tool for time-lapse imaging of microvascular network growth. Previous work in our laboratory has established the use of our model for 1) angiogenesis8, 2) lymphangiogenesis8, 3) pericyte-endothelial cell interactions8, and 4) anti-angiogenic drug testing9. The ability for imaging cultured rat mesentery tissues at multiple time poi...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
DrapeCardinal Health401212” x 12” Bio-Shield Regular Sterilization Wraps
Scalpel HandleRoboz Surgical InstrumentRS-9843Scalpel Handle, #3; Solid; 4" Length
Sterile Surgical BladeCincinnati Surgical0110Stainless Steel; Size 10
Culture Dish (60 mm)Thermo Scientific13018110/Sleeve
Graefe Forcep (curved tweezers)Roboz Surgical InstrumentRS-5135Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4" Length
Graefe Forcep (straight tweezers)Roboz Surgical InstrumentRS-5130Micro Dissecting Forceps; Serrated, Straight; 0.8 mm Tip Width; 4" Length
Noyes Micro ScissorRoboz Surgical InstrumentRS-5677Noyes Micro Dissecting Spring Scissors;
Straight, Sharp-Blunt Points; 13 mm Cutting Edge; 0.25 mm Tip Width, 4 1/2" Overall Length
Gauze PadsFisherBrand13-761-52Non-Sterile Cotton Gauze Sponges; 4" x 4" 12-Ply
Cotton-Tippled ApplicatorsFisherBrand23-400-1246" Length; Wooden Shaft; Single Use Only
6-Well PlateFisher Scientific08-772-49Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic
Sterile Syring 5 mLFisher Scientific14-829-45Luer-Lok Tip
Sterile BowlMedical Action Industries Inc.0123232 oz. Peel Pouch; Blue; Sterile Single Use
6-Well Plate Inserts (CellCrown Inserts)SIGMAZ681792-3EA6-Well Plate Inserts; Non-Sterile
Polycarbonate Filter MembraneSIGMATMTP04700Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0 µm, 47 mm, White Plain
NameCompanyCatalog NumberComments/Description
BeuthanasiaSchering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)MWI #: 011168Active Ingredient: Per 100 mL, 390 mg pentobarbital sodium, 50 mg phenytoin sodium 
KetamineFort Dodge Animal Health (Ordered from MWI Veterinary Supply)MWI #: 000680Kateset 100 mg/mL
XylazineLLOYD. Inc. (Ordered from MWI Veterinary Supply)MWI #: 000680Anased 100 mg/mL
SalineBaxter2F7122
PBSInvitrogen14040-133
MEMInvitrogen11095080
PenStrepInvitrogen15140-122
FBSInvitrogen16000-044
BSAJackson ImmunoResearch001-000-162
Saponin SIGMAS7900-100G
Isopropyl AlcoholFisher ScientificS25372
Povidone-IodineOperand82-226
Hydrochloric AcidSIGMA320331
MethanolFisher Scientific67-56-1
GlycerolFisher Scientific56-81-5
FITC-conjugated LectinSIGMAL9381-2MG
Anti-NG2 Chondroitin Sulfate Proteoglycan AntibodySIGMAAB5320
PECAM (CD31) AntibodyBD Biosciences555026
LYVE-1 AntibodyAngioBio Co.11-034
Goat Anti-Rabbit Cy2-conjugated AntibodyJackson ImmunoResearch111-585-144
Goat Anti-Mouse Cy3-conjugated AntibodyJackson ImmunoResearch115-227-003
Streptavidin Cy3-conjugated AntibodyJackson ImmunoResearch016-160-084
Live/Dead Viability/Cytotoxicity KitInvitrogenL3224
Normal Goat Serum Jackson ImmunoResearch005-000-121
5-Bromo-2'-DeoxyuridineSIGMAB5002
Monoclonal Mouse Anti-Bromodeoxyuridine                        Clone Bu20aDakoM074401-8
Mouse Anti-Rat CD11b AbD SerotecMCA275R

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Tags

Ex Vivo CultureRat Mesentery TissueCapillary SproutingEndothelial Cell ProliferationBSI Lectin LabelingLymphatic Vessel ImagingAngiogenic ResponseFluorescent Microscopy
An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks
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