An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Summary

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Abstract

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Introduction

DR is a serious ocular complication of diabetes, a disease that has reached enormous proportions in the last three decades¹. Twenty years after diagnosis, virtually every patient with type 1 diabetes and 60% of patients with type 2 diabetes present signs of retinopathy, making diabetes per se one of the leading causes of blindness in working age adults². According to the level of microvascular degeneration and ischemic damage, DR is classified into non-proliferative DR (non-PDR) and proliferative DR (PDR). The end-stage disease, PDR, is characterized by ischemia- and inflammation-induced neovascularization and fibrotic responses at the vitreoretinal interface. In untreated conditions, these processes will lead to blindness due to vitreous hemorrhage, retinal fibrosis, tractional retinal detachment, and neovascular glaucoma^3,4. Despite recent advances, current treatment options target only DR stages, including diabetic macular edema and PDR, when retinal damage has already ensued. Moreover, a great proportion of DR patients does not benefit from current treatment armamentarium, indicating an urgent need for improved therapies^4,5,6.

Multiple other in vivo disease/developmental models and diabetic animal models have been developed to date, but none of them recapitulates the full range of pathologic features observed in human PDR^7,8. Moreover, increasing evidence indicates that treatment responses are tightly connected to the ECM composition as well as the spatial arrangement and interaction between the cellular and acellular microenvironment⁹. We, therefore, set out to develop a clinically relevant model of human PDR by utilizing the FT pathological material that is commonly excised from eyes undergoing vitrectomy as part of the surgical management of PDR¹⁰.

This manuscript describes the protocol for the 3D ex vivo culture and characterization of the surgically-excised, PDR patient-derived pathological FT. The method described here has been used in a recent publication that demonstrated successful deconstruction of the native 3D PDR tissue landscape, and recapitulation of features of PDR pathophysiology including angiogenic and fibrotic responses of the abnormal vascular structures¹¹. This model also revealed novel features that cannot be easily appreciated from thin histological sections, such as spatially confined apoptosis and proliferation as well as vascular islet formation¹¹. Vitreous fluid has been successfully used by others on 3D endothelial spheroid cultures to evaluate its angiogenic potential and the efficacy of angiostatic molecules¹². When combined with an in vitro 3D lymphatic endothelial cell (LEC) spheroid sprouting assay using PDR vitreous as stimulant, our model revealed the contribution of both soluble vitreal factors as well as local cues within the neovascular tissue to the as yet poorly understood LEC involvement in PDR pathophysiology^3,11. In the management of PDR, vitreoretinal surgery is a routinely performed yet challenging procedure. As surgical instrumentations and techniques are seeing continuous advancement and sophistication, timely and conservative removal of fibrovascular proliferative specimen not only improves vision outcome but also provides invaluable tissue material for the investigation of PDR pathophysiology and treatment responses in the complex translational aspects of the live human tissue microenvironment.

Protocol

This research was approved by the Institutional Review Board and Ethical committee of Helsinki University Hospital. Signed informed consent was obtained from each patient.

1. Preparation of Solutions, Media and Equipment

1. Prepare the following equipment prior to collection of the fibrovascular tissue (FT) to ensure rapid processing.
   1. Sterile-autoclave two microdissection tweezers.
   2. Prepare 1x phosphate-buffered saline (PBS) by dissolving 1 pre-weighed PBS tablet (0.14 M NaCl, 0.0027 M KCl, 0.010 M PO₄^-3) in 900 mL of deionized water and stir to dissolve. Adjust the water volume up to 1 L and sterile-autoclave the ready solution. Store at RT.
   3. Gather the above mentioned solution and equipment in a basket, together with a single-use sterile scalpel, a sterile 6-cm cell culture dish and five sterile 12-well cell culture plates.
2. Prepare a 6 mg/mL fibrinogen solution in Hanks Balanced Salt Solution (HBSS) by dissolving 30 mg of plasminogen-depleted human fibrinogen into 5 mL of HBSS, using a 50 mL tube immersed into a water bath at 37 °C, for at least 1 h.
   NOTE: This solution can be kept in the water bath (37 °C) for 7 h (maximum 10 h) before use.
3. Prepare the ex vivo culture medium by adding 5% fetal calf serum (FCS) or 5% human serum, 0.4% endothelial cell growth supplement, 10 ng/mL recombinant human epidermal growth factor, 1 μg/mL hydrocortisone, and 50 μg/mL gentamycin to commercially available endothelial cell growth medium and keep in water bath at 37 °C until use. Store at 4 °C for up to a month.

2. Fibrovascular Tissue Dissection

1. Collect the fibrovascular tissue (FT) that has been excised from human subjects undergoing trans-conjunctival microincision vitreoretinal surgery, by 23 or 20 gauge three-port pars plana vitrectomy as part of the vitreoretinal surgical management of PDR.
   NOTE: The FT is excised using segmentation and delamination, cut if needed with microscissors, and removed from the vitreous cavity with intraocular end-gripping microforceps by experienced vitreoretinal surgeon. Extreme care needs to be taken to remove intact, undamaged FT without causing damage to ocular structures including the optic nerve or temporal vascular arcade where the pathological neovascular tissue is most commonly located. The size of the excised tissue is variable, usually ranging between 3 and 50 mm².
2. Keep the FT in a 6 cm cell culture dish or 1.5 mL tube containing 1 mL of sterile PBS placed on wet ice, and transfer it immediately to research laboratory for processing.
   NOTE: It is essential that the research unit (lab facilities) are physically close to the hospital operating room (OR) to minimize the transfer times of the small excised FT. In this case the transfer takes less than 5 minutes and the explants are embedded within fibrin usually in 1 - 1.5 h (maximum 2 h) after surgical removal. The impact of longer transfer times on the 3D culture would need to be tested.
3. Place the FT in a 6-cm cell culture dish containing 1 mL of PBS at room temperature (RT, 25 °C) and acquire images of the tissue using an inverted epifluorescence microscope with a 5x objective.
   NOTE: Images are acquired for qualitative assessment and documentation. It will be possible to observe the tissue size, density and general composition (e.g., vascular structures).
4. Cut the FT into ~1 mm² pieces under an upright dissection stereomicroscope, using a sterile scalpel and microdissection tweezers. Hold the tissue, well submerged in PBS, in place using a microdissection tweezer, and perform clear cuts using a sterile scalpel. Avoid tearing FT, as this would alter the native fibrovascular structures.
5. Place each individual piece into a well of a 12-well cell culture plate containing 1 mL of sterile PBS.
   NOTE: If needed, gently spread the FT onto the plate, using microdissection tweezers, prior to performing cuts.

3. Casting Upright Fibrin Gel Droplets for the Characterization of Native FT and Ex Vivo Culture

1. Sterile-filter the ready fibrinogen solution prepared in step 1.2 with a 0.22 μm filter mounted into a 10 mL syringe.
2. Prepare aliquots of the fibrinogen solution (25 μL per 1.5 mL tube) and keep at RT. Prepare an aliquot for fibrin gel/every FT piece obtained after the dissection, and a couple of extra aliquots for testing the fibrin gel formation.
3. Prepare a solution of HBSS containing 4 units/mL of thrombin and 400 μg/mL of aprotinin, called hereafter TA solution, and keep at RT. Prepare as much TA solution as needed, depending on the number of pieces obtained after dissection (25 μL of TA solution is used for each FT/fibrin gel), and an extra amount (e.g., 250 μL) for testing the fibrin gel formation and ensuring that enough solution is available throughout casting of the fibrin gel droplets.
   NOTE: Thrombin is required for fibrin polymerization while aprotinin is added to prevent degradation of the fibrin gel by cellular proteases expressed by the FTs^13,14.
4. Test the timing of fibrin gel formation: add 25 μL of TA solution to the aliquoted 25 μL fibrinogen solution prepared in step 3.2 and, using another pipette set to 50 μL, mix in the tube by pipetting and dispense into a 6 cm cell culture dish, forming an upright droplet.
   NOTE: The fibrin gel formation should take place in ~1 min. If this takes over 1.5 min, the concentration of thrombin in the TA solution prepared in step 3.3 can be increased by adding more thrombin stock solution. One tenth of the initially used volume of thrombin stock solution can be added, repeatedly, until the fibrin gel formation occurs within 1.5 min. The time of fibrin gel formation is critical for the three dimensionality of the culture, as quick gel formation (within 1.5 minutes) prevents the FT piece from sinking to the bottom of the fibrin gel, and thus coming in contact with the 2D plastic surface of the cell culture plate, which can lead to 2D cell adhesion and outgrowth.
5. Place one FT piece at the center of one well of a 24-well plate using a sterile tweezer and remove any excess PBS by pipetting with a 0.5-10 μL pipette to avoid suction force on the FT. In the case the tissue sticks to the tweezer, use an additional tweezer to aid the placement of the tissue piece on the plate.
   NOTE: FT/fibrin gels can also be cast on 48-well plate to reduce the usage of reagents. However, this is more challenging as the space is more limited and fibrin will spread if it comes into contact with the well wall.
6. Add 25 μL of TA solution to the 25 μL fibrinogen solution aliquoted in step 3.2, and using another pipette set to 50 μL mix in the tube by pipetting. Dispense onto the FT piece placed on the plate well, and pipette up and down 2- 3 times in order to include the FT piece within the upright droplet, providing a 3D surrounding matrix to the FT.
   NOTE: Ensure that the FT is not aspirated during pipetting and that no air bubbles are formed. Ensure also that mixing, dispensation and pipetting are performed quickly as the fibrin gel will form within 1.5 min, as tested in step 3.4. If casting fibrin gels on 48-well plate, use instead 20 μL of fibrinogen solution and 20 μL of TA solution.
7. Repeat steps 3.5 and 3.6 for all the FT pieces.
8. Allow the fibrin gels to solidify by incubating the plates in a cell culture incubator at 37 °C in a humidified atmosphere with 5% CO₂.
9. After 0.5-1 h, check that the fibrin gel is completely formed by carefully tilting the plate. Proceed to step 3.10 when the gel does not move as the plate is tilted, indicating that the fibrin gel formation is complete.
10. Overlay the FT/fibrin gels with ex vivo culture medium (600 μL/well in 24-well plate or 300 μL/well in 48-well plate) supplemented with 100 μg/mL aprotinin. Pipette the medium gently by drop-wise dispensation.
    NOTE: Here, aprotinin is used to prevent degradation of the fibrin gel by cellular proteases expressed by the FTs^13,14. The ex vivo culture medium can additionally be supplemented with recombinant growth factors, such as VEGFA, VEGFC, bFGF, TGFβ (see Table of Materials)¹¹.
11. Place the FT ex vivo culture plate back into the 37 °C, 5% CO₂ cell culture incubator and culture for the desired time period (e.g., 2- 18 days, longer culture periods will need to be tested). Replace 50% of the medium with fresh ex vivo culture medium every three days.

4. “In Matrix” Imaging

1. Acquire images of the FT ex vivo cultures on the same day (day 0) and at set intervals (e.g., every other day), using an inverted epifluorescence microscope, in order to follow the 3D growth.
   NOTE: The images obtained can be used for the quantification of the FT outgrowth as the total length of two perpendicular diameters across the explant¹¹.

5. Native FT and Ex Vivo Culture End-Point

NOTE: The FT/fibrin gels can be cultured ex vivo for the desired time period (FT ex vivo cultures) or fixed on the same day (native FT) for native FT characterization¹¹.

1. Fix the native FT or the FT ex vivo cultures by replacing the culture medium with 1 mL/well of 4% paraformaldehyde in PBS solution, for 1 h at RT. For the native FT/fibrin gels, fix 0.5-1 h after addition of the ex vivo culture medium (if the fibrin gels have not been soaked in medium, fixation will damage them).
   NOTE: Perform this step in fume hood as paraformaldehyde is corrosive, toxic and carcinogenic.
2. Rinse the FT/fibrin gels with three 5 min washes in PBS (2 mL/well).
3. Replace the PBS with 2 mL/well of PBS containing 0.02% sodium azide and store the fixed fibrin gels at 4 °C until further processing. Seal the plates with paraffin film to ensure that the FT/fibrin gels do not dry in storage.
   NOTE: Perform this step in fume hood as sodium azide is toxic.

6. Whole-Mount Immunofluorescence Staining

NOTE: This protocol lasts 5 days and can be interrupted with overnight incubations where indicated. Do not let the fibrin gels dry at any point. All steps are performed at RT unless otherwise indicated.

1. Prepare the following solutions before starting the staining protocol.
   1. Post-fixation solution: prepare 50:50 acetone:methanol solution by mixing equal amounts of acetone and methanol (e.g., 50 mL). Store at -20 °C. Prepare this solution latest on the day before the staining. This solution can be stored back at -20 °C and be used for subsequent stainings.
      NOTE: Prepare this solution in fume hood as acetone and methanol are flammable and hazardous to health.
   2. Blocking solution: prepare a 15% fetal bovine serum (FBS), 0.3% octyl phenol ethoxylate solution in PBS by first adding 15% FBS to PBS. Add octyl phenol ethoxylate and stir to dissolve. Store at 4 °C.
      NOTE: This solution can be prepared in large amount in advance, stored in 15 mL aliquotes at -20 °C and thawed before use, on the day when the staining protocol is started. Use a freshly prepared or freshly thawed aliquote for each staining protocol.
   3. Washing solution: Prepare 1 L of PBS supplemented with 0.45% octyl phenol ethoxylate by adding 4.5 mL of octyl phenol ethoxylate to 995.5 mL of PBS. Stir to dissolve. Store at RT.
2. Lift the droplets carefully from the plate by using a stainless steel square-edged spatula. Detach the droplet first from the edges before lifting the center, and transfer to a well of 12-well plate containing 1 mL of PBS.
   NOTE: Perform post-fixation, washes and blocking steps in this plate format.
3. Post-fix the droplets with 1 mL of ice-cold 50:50 acetone:methanol solution for ~1 min (the border of the droplets will turn white).
   NOTE: Perform this step in fume hood as acetone and methanol are hazardous to health.
4. Rinse with 3 - 5 washes in 2 mL of PBS (the droplets will sink in the PBS solution when rehydration is complete).
   NOTE: Avoid touching the droplets with the pipette tip as, after post-fixation, they are sticky to plastic. Throughout the protocol, avoid aspirating post-fix, antibody, blocking and washing solutions by suction, as this may damage or completely destroy the droplets. Instead, tilt the plate and carefully remove solutions using a 500-5,000 μL pipette.
5. Centrifuge the blocking solution for 15 min at 21,000 x g and 4 °C in order to remove debris.
   NOTE: The solution can be aliquoted in 1.5 mL tubes for centrifugation. The unused solution can be stored at 4 °C and used for all relevant subsequent steps.
6. Incubate the droplets in 500 μL blocking solution for 2 h at RT.
   NOTE: To reduce the volume of blocking solution used, the plate can be tilted on a support and as little as 300 μL of solution/droplet can be used.
7. Prepare the primary antibody mixture (at least 30 μL/droplet) at appropriate dilution in blocking solution and centrifuge for 15 min at 21,000 x g and 4 °C.
8. Transfer the droplets into round (U)-bottom 96-well plate by using a round-edged spatula, and incubate with at least 30 μL/droplet of the primary antibody mixture overnight at 4 °C.
9. On the following day, transfer the droplets into 12-well plate containing 2 mL of washing solution/well, rinse with three 5 min washes first and then with nine 30 min washes. Leave the last wash (2 mL) overnight at 4 °C.
10. On the following day, rinse three times with PBS and transfer the droplets into round (U)-bottom 96-well plate.
11. During the washes, prepare the appropriate fluorophore-conjugated secondary antibody mixture (diluted 1:500, at least 30 μL/droplet) in blocking solution and centrifuge for 15 min at 21,000 x g and 4 °C.
12. Transfer the droplets into a round (U)-bottom 96-well plate by using a round-edged spatula and incubate with at least 30 μL of the secondary antibody mixture, for 4 h at RT, protected from light.
    NOTE: Perform all subsequent incubations and washing steps protected from light.
13. Transfer the droplets into 12-well plate containing 2 mL of washing solution/well using a round-edged spatula, rinse with three 5 min washes first and then with four 30 min washes. Leave the last wash (2 mL) overnight at 4 °C.
14. On the following day, rinse the gels with five 30 min washes and then three times with PBS. Leave the last wash (2 mL) overnight at 4 °C.
15. On the following day, transfer the droplets into a round (U)-bottom 96-well plate by using a round-edged spatula and counterstain nuclei by incubating with 10 μg/mL Hoechst nuclear stain (at least 30 μL/droplet) for 30 minutes at RT.
    NOTE: Mounting medium supplemented with 4',6-diamidino-2-phenylindole (DAPI) for nuclei counterstaining can alternatively be used. In which case, this step and step 6.16 are skipped, proceeding directly to step 6.17.
16. Transfer the droplets into 12-well plate containing 2 mL of PBS/well using a round-edged spatula and rinse three times with PBS.
17. Mount the droplets onto microscope slide as follows:
    1. Apply a narrow layer of quick-hardening mounting medium on the edges of a square-shaped 22 mm x 22 mm coverglass and let dry for ~1 minute. Perform this step for each drop individually, to avoid excessive hardening of the mounting medium.
       NOTE: Perform this step in fume hood as the quick-hardening mounting medium is hazardous to health.
    2. Rinse the droplet by dipping in a well of a 12-well plate containing 2 mL of deionized water and transfer onto a microscope slide, using a round-edged spatula. Remove excess water by using a small piece of absorbent paper.
    3. Dispense 15 μL of non-hardening antifade mounting medium onto the droplet.
       NOTE: Alternatively, if Hoechst nuclear stain has not been used, mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) can be used.
    4. Gently position the cover glass, with the quick-hardening mounting medium facing the microscopic slide, over the droplet and let settle.
       NOTE: The quick-hardening medium will stick to the microscopic slide and keep the droplet in place.
18. Repeat step 6.17 for all droplets. Mount two droplets on each microscope slide.
19. Let the slides air-dry at RT for ~2 h and store at 4 °C overnight. Ensure that the slides are positioned horizontally and protected from light.
20. Image the stained native or end-point FT ex vivo cultures using a confocal microscope or an upright epifluorescence microscope equipped with an optical sectioning function and 20x or 40x objective. Use tile function to capture a greater area of the tissue at once.

Results

Deeper understanding of the PDR fibrovascular tissue properties and protein expression has relied mainly on vitreous samples and thin histological FT sections^3,15,16,17. To develop a method for thorough investigation of the 3D tissue organization and multicellular physiopathological processes of PDR, we set out to utilize the surgically excised, patient-derived ...

Discussion

Considering the importance of relevant tissue microenvironment for reliable functional cell and molecular mechanistic results, it is imperative to find appropriate experimental models that provide this tissue environment. The herein described ex vivo PDR culture model for the fibrin-embedded FTs allows the investigation of the mechanisms of PDR pathophysiology in the native, complex and multicellular context of the PDR clinical samples.

Critical steps within the protocol are the prope...

Materials

Name	Company	Catalog Number	Comments
Material
Microforceps	Medicon	07.60.03	Used for handling the FTs
Disposable Scalpels - Sterile	Swann-Morton	0513	Used for FT dissection
Culture dish, vented, 28 ml (60mm)	Greiner Bio-One	391-3210	Used for dissection and for testing fibrin gel formation
Cell culture plates, 12-well	Greiner Bio-One	392-0049	Used for FT dissection and whole-mount immunofluorescence
Reagent/centrifuge tube with screw cap, 15 mL	Greiner Bio-One	391-3477
Reagent/centrifuge tube with screw cap, 50 mL	Greiner Bio-One	525-0384
Millex-GV Syringe Filter Unit, 0.22 µm, PVDF	Millipore	SLGV033RS	Used to sterile-filter the fibrinogen solution
Syringe, 10 mL	Braun	4606108V	Used to sterile-filter the fibrinogen solution
Polypropylene Microcentrifuge Tubes, 1.5 mL	Fisher	FB74031
Cell-Culture Treated Multidishes, 24-well	Nunc	142475	Used for casting the FT/fibrin gels for native FT characterization and ex vivo culture
Cell culture plates, 96-well, U-bottom	Greiner Bio-One	392-0019	Used for whole-mount immunofluorescence
Round/Flat Spatulas, Stainless Steel	VWR	82027-528	Used for whole-mount immunofluorescence
Coverslips 22x22mm #1	Menzel/Fisher	15727582	Used for mounting
Microscope slides	Fisher	Kindler K102	Used for mounting
Absorbent paper	VWR	115-0202	Used for mounting
Name	Company	Catalog Number	Comments
Reagents
PBS tablets	Medicago	09-9400-100	Used for preparing 1x PBS
Fibrinogen, Plasminogen-Depleted, Human Plasma	Calbiochem	341578
Hanks Balanced Salt Solution	Sigma-Aldrich	H9394-500ML	Used for preparing the fibrinogen and TA solution
Fetal bovine serum	Gibco	10270106	Used for preparing the blocking solution
Human Serum	Sigma-Aldrich	H4522	Aliquoted in -20 °C, thaw before preparing the ex vivo culture media
Gentamicin Sulfate 10mg/ml	Biowest	L0011-100
Endothelial cell media MV Kit	Promocell	C-22120	Contains 500 ml of Endothelial Cell Growth Medium MV, 25 mL of fetal calf serum, 2 mL of endothelial cell growth supplement,  500 μL of recombinant human epidermal growth factor (10 μg/ mL) and 500 μL of hydrocortisone (1 g/ mL)
Sodium azide	Sigma-Aldrich	S2002	Used for storage of the native and ex vivo cultured FTs. TOXIC: wear protective gloves and/or clothing, and eye and/or face protection. Use in fume hood.
Acetone	Sigma-Aldrich	32201-2.5L-M	Used to prepare the post-fixation solution. HARMFUL: wear protective gloves and/or clothing. Use in fume hood.
Methanol	Sigma-Aldrich	32213	Used to prepare the post-fixation solution. TOXIC: wear protective gloves and/or clothing. Use in fume hood.
Triton X-100 (octyl phenol ethoxylate)	Sigma-Aldrich	T9284	Used for whole-mount immunofluorescence. HARMFUL: wear protective gloves and/or clothing.
Hoechst 33342, 20mM	Life Technologies	62249	For nuclei counterstaining. HARMFUL: wear protective gloves and/or clothing, and eye and/or face protection.
VECTASHIELD Antifade Mounting Medium	Vector Laboratories	H-1000	Wear protective gloves and/or clothing, and eye protection. Use in fume hood.
VECTASHIELD Antifade Mounting Medium with DAPI	Vector Laboratories	H-1200	Mounting medium with nuclei counterstaining. Wear protective gloves and/or clothing, and eye protection. Use in fume hood.
Eukitt Quick-hardening mounting medium	Sigma-Aldrich	03989-100ml	TOXIC: Wear protective gloves and/or clothing, and eye protection. Use in fume hood.
Thrombin from bovine plasma, lyophilized powder	Sigma-Aldrich	T9549-500UN	Dissolve at 100 units/ mL, aliquote and store at -20 °C, avoid repeated freeze/ thaw
Aprotinin from bovine lung, lyophilized powder	Sigma	A3428	Dissolve at 50 mg/ mL, aliquote and store at -20 °C, avoid repeated freeze/ thaw
Name	Company	Catalog Number	Comments
Growth factors
Recombinant human VEGFA	R&D Systems	293-VE-010	50 ng/ mL final concentration
Recombinant human VEGFC	R&D Systems	752-VC-025	200 ng/ mL final concentration
Recombinant human TGFβ	Millipore	GF346	1 ng/ mL final concentration
Recombinant human bFGF	Millipore	01-106	50 ng/ mL final concentration
Name	Company	Catalog Number	Comments
Primary antibodies
CD31 (JC70A)	Dako	M0823	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
CD34 (QBEND10)	Dako	M716501-2	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
CD45 (2B11+PD7/26)	Dako	M070129-2	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
CD68	ImmunoWay	RLM3161	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
Cleaved caspase-3 (5A1E)	Cell Signalling	9664	Used at 1:200 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
ERG (EP111)	Dako	M731429-2	Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
GFAP	Dako	Z0334	Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
Ki67	Leica Microsystems	NCL-Ki67p	Used at 1:1500 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
Lyve1	R&D Systems	AF2089	Used at 1:100 dilution, Donkey anti Goat Alexa 568 Secondary Ab
NG2	Millipore	AB5320	Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
Prox1	ReliaTech	102-PA32	Used at 1:200 dilution, Goat anti Rabbit Alexa 568 Secondary Ab
Prox1	R&D Systems	AF2727	Used at 1:40 dilution, Chicken anti Goat Alexa 594 Secondary Ab
VEGFR3 (9D9F9)	Millipore	MAB3757	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
α-SMA (1A4)	Sigma	C6198	Used at 1:400 dilution, Cy3 conjugated
Name	Company	Catalog Number	Comments
Secondary antibodies
Alexa Fluor488 Donkey Anti-Mouse IgG	Life Technologies	A-21202	Used at 1:500 dilution
Alexa Fluor594 Goat Anti-Rabbit IgG	Invitrogen	A-11012	Used at 1:500 dilution
Alexa Fluor568 Donkey anti-Goat IgG	Thermo Scientific	A-11057	Used at 1:500 dilution
Alexa Fluor568 Goat anti-Rabbit IgG	Thermo Scientific	A-11036	Used at 1:500 dilution
Alexa Fluor594 Chicken Anti-Goat IgG	Molecular Probes	A-21468	Used at 1:500 dilution
Name	Company	Catalog Number	Comments
Microscopes
Axiovert 200 inverted epifluorescence microscope	Zeiss		For imaging of the fresh and fibrin-embedded FT
SZX9 upright dissection stereomicroscope	Olympus		For FT dissection
LSM 780 confocal microscope	Zeiss		For imaging of whole-mount immunostained FT
AxioImager.Z1 upright epifluorescence microscope with Apotome	Zeiss		For imaging of whole-mount immunostained FT