WO2004111207A1

WO2004111207A1 - Multicellular in vitro model of angiogenesis and methods for using the same

Info

Publication number: WO2004111207A1
Application number: PCT/EP2004/007198
Authority: WO
Inventors: Mark Nesbit
Original assignee: Centelion
Priority date: 2003-06-09
Filing date: 2004-06-09
Publication date: 2004-12-23

Abstract

The invention relates to a multicellular in vitro composition comprising (A) at least one spheroid of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix, and (C) a serum-free growth medium. The composition is capable of expressing angiogenic growth characteristics, for example, endothelial cell lumenization and branching. The invention also relates to methods of using the composition as an in vitro model of angiogenesis, to assay and screen for compounds that modulate angiogenesis, and to study and research angiogenesis. The invention further relates to kits comprising the composition, and methods for making the composition.

Description

MULTICELLULAR IN VITRO MODEL OF ANGIOGENESIS AND METHODS FOR USING THE SAME

Field of the Invention and Introduction

The invention provides, for the first time, an in vitro model of angiogenic endothelial cell lumenization and branching. This model is based on using an in vitro multicellular composition comprising at least one spheroid of endothelial cells and smooth muscle cells, a fibrin matrix, and a serum-free growth medium.

Background of the Invention

In vivo, the two main vascular cell types are vascular endothelial cells and mural cells. Vascular endothelial cells form a monolayer throughout the entire vasculature as polarized cells with an apical surface (towards vessel lumen) and a basal surface (towards "outside"), which is surrounded by a basal lamina (or basement membrane). Mural cells wrap around this structure and are contractile cells that regulate vessel diameter and blood flow. Mural cells on large vessels are multi-layered and known as smooth muscle cells. On smaller vessels, such as capillaries, mural cells are more sparse and usually known as pericytes.

The process whereby new vessels originate as capillaries, which sprout from existing small vessels, is called angiogenesis. Angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. The control of angiogenesis is a highly regulated and complex system involving extensive interplay between angiogenic stimulators and inhibitors, cell types, soluble factors, and extracellular matrix components. (Liekens et al., Angiogenesis: regulators and clinical applications, Biochem Pharmacol 61 :253-270, 2001.) Thus, angiogenesis is a central component of the body's normal physiology, for example, during wound healing. In addition, the control of angiogenesis has been found to be altered in certain disease states, and, in many cases, the pathological damage associated with the disease is related to the uncontrolled angiogenesis. One detrimental aspect, for example, occurs when blood vessels multiply and enhance growth and metastasis of tumors. Aberrant angiogenesis is also associated with numerous disorders, including rheumatoid arthritis, where blood vessels invade the joint and destroy cartilage, and numerous ophthalmologic pathologies, such as diabetic retinopathies in which new capillaries invade the vitreous, bleed and cause blindness, and macular degeneration, prostate cancer and Kaposi's carcinoma. Angiogenesis is essential to tumor development and growth. Prevention of angiogenesis can inhibit solid tumor growth. Compounds that have anti-angiogenic activity can be used to treat disorders or disease involving abnormally excessive angiogenesis.

Compounds that have anti-angiogenic activity can also be used, for example, as anti-tumor agents and for the treatment of ophthalmic disorders, particularly involving the retina and vitreous humor, and for hyperproliferative dermatological disorders, such as psoriasis, that have an angiogenic component. Thus, compounds that promote angiogenesis and compounds that inhibit angiogenesis are being sought. This has led to a search for specific inhibitors of endothelial cell growth. As a result, there is an interest in measuring proliferation of endothelial cells under inhibitory and stimulatory conditions as screens for discovery of inhibitors (or alternatively stimulators) of angiogenesis.

Models of angiogenesis have been developed and used as screens for discovery of inhibitors or stimulators of angiogenesis. Assays using ex vivo endothelial tissue have had the advantage of providing a model of endothelial tube formation and branching in actual vascular tissues. Such ex vivo assays, however, involve time-consuming microsurgical preparation, and because consecutive samples lack uniformity, results are inconsistent because of the difficulty in quantifying differences between samples. In vitro assays, on the other hand, provide improved homogeneity between samples and improved consistency in quantification techniques. Inconsistent or inadequate endothelial tube formation and branching, however, limits quantification of meaningful morphological changes in these in vitro models. Consequently, direct assessment of angiogenesis models, such as by quantifying endothelial tube formation and branching, has been replaced by indirect methods, such as by packed cell volume, by chemical determination of a cellular component, for example, protein or deoxyribonucleic acid, or by uptake of a chromogenic dye such as neutral red. Thus, there is a need for convenient and reproducible assays for identifying agents that modulate angiogenesis and for quantifying their potential to modify angiogenesis. Accordingly, it is an object herein to provide a method for identifying compounds that modulate angiogenesis by incorporating the advantages of models known in the art and mitigating or obviating their corresponding disadvantages. Therefore, it is also an object herein to provide a model of angiogenesis comprising the advantages of quantifiable morphological changes of ex vivo models (such as, for example, endothelial tube formation and branching) and/or the advantages of in vitro models including improved homogeneity between samples and improved consistency in quantifying results.

The object of the present invention is to provide an in vitro model of angiogenesis which encompasses quantifiable changes, such as, for example, endothelial tube formation and branching, improved homogeneity between samples, improved consistency in quantifying results, and/or an in vitro model that can be used to examine both stimulation and inhibition of angiogenesis. For example, the present invention provides an in vitro assay of angiogenesis which encompasses quantifiable changes, such as, for example, endothelial tube formation and branching.

One aspect of the present invention relates to a multicellular in vitro composition that expresses quantifiable morphological changes due to angiogenesis within the composition, such as, for example, endothelial tube formation and branching. The composition generally comprises (A) at least one spheroid of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix, and (C) a serum-free growth medium. Numerous coculture systems of endothelial cells (EC) and smooth muscle cells (SMC) have been developed to study paracrine interactions in the vessel wall. These include planar coculture models of cells cultured together in the same dish, bilayer coculture, two-compartment filter systems, and agarose cocultures (Fillinger, M. F. et al. (1997) Coculture of endothelial cells and smooth muscle cells in bilayer and conditioned media models. J. Surg. Res. 67,169-178; Bonin, L. R, Damon, D. H. (1994) Vascular cell interactions modulate the expression of endothelin-1 and platelet-derived growth factor BB. Am. J. Physiol. 267,H1698-H1706; Powell, R. J., Bhargava, J., Basson, M. D., Sumpio, B. E. (1998) Coculture conditions alter endothelial modulation of

TGF-beta 1 activation and smooth muscle growth morphology. Am. J. Physiol. 274,H642-H649; Hirschi, K. K., Rohovsky, S. A., D'Amore, P. A. (1998) platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and heterotypic cell-cell interactions mediate endothelial cell- induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J. Cell Biol. 141,805-814; Axel, D. I., Riessen, R., Athanasiadis, A., Runge, H., Koveker, G, Karsch, K. R. (1997) Growth factor expression of human arterial smooth muscle cells and endothelial cells in a transfilter coculture system. J. Mol. Cell. Cardiol. 29,2967-2978.) These studies have shown that EC and SMC regulate each other's quiescent phenotype. EC- derived PDGF-BB controls mural cell recruitment and differentiation (Lindahl, P., Johansson, B. R., Leveen, P., Betsholtz, C. (1997) Pericyte loss and microaneurysm formation in PDGF-B -deficient mice. Science 277,242-245; Hirschi, K. K., Rohovsky, S. A., D'Amore, P. A. (1998).) In turn, mural cell- derived, activated transforming growth factor β (TGF-β) contributes to the maintenance of the quiescent EC phenotype (Antonelli-Orlidge, A., Saunders, K. B., Smith, S. R., D'Amore, P. A. (1989) An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes. Proc. Natl. Acad. Sci. USA 86,4544-4548; Sato, Y., Rifkin, D. B. (1989) Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture. J. Cell Biol. 109,309-315.) Contact of endothelial cells with mural cells (pericytes, smooth muscle cells) has been shown to limit a plasticity window for vessel remodeling and to render endothelial cells independent of the activities of survival factors such as VEGF, FGF-2, or Ang-1 (Benjamin, L., Hemo, I., Keshet, E. (1998) A plasticity window for blood vessel remodeling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and vascular epithelial growth factor (VEGF). Development 125,1591-1598; Alon, T., Hemo, I., Itin, A., Pe'er, J., Stone, J., Keshet, E. (1995) Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat. Med. 1,1024-

1028.)

Spheroids of endothelial cells (EC) and smooth muscle cells (SMC) on a collagen matrix and in a serum-containing growth medium have recently been shown to differentiate spontaneously to organize into a core of SMC and a surface layer of EC, that is to be capable of mimicking the 3-dimensional assembly of a blood vessel with a luminal aspect, a polarized endothelial cell monolayer, and an underlying multilayered assembly of smooth muscle cells. (Korff et al, FASEB J, 15:447-457 (2001), incorporated herein by reference.) Furthermore, it has been shown by Darland et al., Curr. Top.Dev.Biol. 52, 107-149) that SMC inhibit EC proliferation and new vessel formation in cocultures, implying that mural cells may interact with EC to stabilize newly formed capillaries. EC cocultured with SMC became refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). (Id.) In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness.

The present inventors have unexpectedly discovered an in vitro spheroid composition that is capable of expressing robust angiogenic morphologies, such as, for example, endothelial tube formation and branching. Expression of these vascular growth structures from the inventive composition is so vigorous and consistent that quantitation of these structures in matched samples generally provides reliable and reproducible results.

While EC/SMC spheroids grown with serum on cellulose matrix are minimally sensitive to VEGF, the present inventive EC/SMC spheroids grown with serum-free medium on a fibrin matrix are many times more sensitive to VEGF. In another embodiment, the EC/SMC spheroids grown with serum- free medium on a fibrin matrix are even more sensitive to VEGF when bFGF is present, resulting in endothelial cell lumenization, such as tube formation, and branching.

Summary of the Invention

The present invention relates to an in vitro composition comprising at least one spheroid of endothelial cells and smooth muscle cells, a fibrin matrix or precursors for a fibrin matrix; and a serum-free growth medium. The present invention also relates to an in vitro composition comprising at least one spheroid of endothelial cells and smooth muscle cells, a fibrin matrix or precursors for a fibrin matrix; a serum-free growth medium, and plasma.

Preferably, the spheroids composition according to the invention has not been exposed to platelets or transforming growth factor, or to platelet degranulation products.

The composition according to the invention comprises endothelial cells are selected from human umbilical vein endothelial cells (HUVEC), or human tissue in general. The composition according to the present invention comprises smooth muscle cells that may derive from pulmonary artery smooth muscle cells

(PASMC), or other human tissue.

The present invention also provides an in vitro composition comprising at least one spheroid of endothelial cells and smooth muscle cells, a fibrin matrix or precursors for a fibrin matrix, which may derive from fibrin precursors are fibrinogen and thrombin. The present invention further provides an in vitro composition comprising at least one spheroid of endothelial cells and smooth muscle cells, a fibrin matrix or precursors for a fibrin matrix; and a serum-free growth medium, wherein the spheroid comprises endothelial cell lumenization structures, branching structures, or combinations thereof.

Another object of the present invention is an in vitro composition comprising at least one spheroid of endothelial cells and smooth muscle cells, a fibrin matrix or precursors for a fibrin matrix; and a serum-free growth medium, and further comprising bFGF, VEGF, or both. Still another object of the present invention is The present invention relates to an in vitro composition comprising at least one spheroid of endothelial cells and smooth muscle cells, a fibrin matrix or precursors for a fibrin matrix; and a serum-free growth medium, further comprising at least one agent that is known to promote or inhibit angiogenesis. The present composition according to the present invention is thus useful for screening an agent for its ability to alter angiogenesis. Such agent may be a low molecular weight small molecule that can be synthesized in vitro. Effective anti-angiogenic agent so screened can then be administered to an animal to prevent or treat disorders or diseases involving abnormal angiogenesis.

The present invention also provides a multicellular in vitro assay method for screening an agent for its ability to alter angiogenesis comprising: preparing an in vitro composition comprising (A) spheroids of endothelial cells and smooth muscle cells, (B) a fibrin matrix, and (C) a serum-free growth medium; preparing separate aliquots of the composition, wherein each aliquot comprises at least one spheroid; adding to a test aliquot an agent to be screened for its ability to alter angiogenesis; maintaining a control aliquot wherein the agent is not added; maintaining the aliquots for a sufficient time under sufficient conditions to allow a spheroid in at least one of the aliquots to express angiogenic features; measuring spheroid morphology in the aliquots at least once after angiogenic features are expressed; comparing the spheroid morphology measurements in the test aliquot to those of the control aliquot; and determining the ability of the agent to alter angiogenesis in the spheroid. The method according to the present invention is therefore useful predicting in vivo outcomes for using the agent to prevent or treat disorders or diseases involving excessive angiogenesis by referring to the measurements of the agent's ability to inhibit angiogenesis in the in vitro spheroid, or by referring to the numbers of tubes or lumens formed in comparison with control untreated spheroids composition.

The present invention further provides with a kit for analyzing the angiogenesis modulating effect of a compound, comprising an in vitro composition comprising (A) spheroids of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix; and (C) a serum-free growth medium, with sufficient nutrients to allow growth of new vascular tissue.

The present invention still further provides with a method of making a multicellular in vitro composition that is capable of expressing in vitro endothelial cell lumenization and braching, comprising combining (A) spheroids of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix; and (C) a serum-free growth medium, with sufficient nutrients and growth factors to allow growth of new vascular tissue. Brief Description of Drawings

Fig. 1. Spheroid formation with HUVEC at day 0.

Fig. 2. Growth of HUVEC spheroids at day 7 with no bFGF/VEGF(A), with bFGF alone (B), with VEGF alone (C), and with both bFGF and VEGF

(D).

Fig. 3. Inhibition of HUVEC spheroid growth by staurosporine (ST -

Staurosporine, synthetic, CAS 62996-74-1, C28H26N403, MW 466.5; was provided by A.G. Scientific Inc. (S-1016)).

Fig. 4. Quantitation of spheroid morphology with Image pro plus

Invert scope with z stack with VEGF 200ng/ml (A) and VEGF 400 ng/ml (B).

Fig. 5. PASMC spheroid at day 1 post implantation in 2% serum (A) and 2% plasma (B).

Fig. 6. Growth of HUVEC spheroids (A) and PASMC spheroids (B) in fibrin/plasma with bFGF and VEGF, at day 2.

Fig. 7. Growth of HUVEC/PASMC spheroids in fibrin/plasma with bFGF and VEGF, at day 7, with various proportion of EC and SMC.

Fig. 8. Quantifying growth of HUVEC/PASMC spheroids in fibrin/plasma with bFGF and VEGF, at day 7.

Fig. 9. Quantifying growth of HUVEC/PASMC spheroids in fibrin/plasma with bFGF and VEGF, at day 7.

Fig. 10. Growth of HUVEC/PASMC spheroids in fibrin/plasma with bFGF and VEGF, at day 4, labeled PASMC, and labeled HUVEC.

Fig. 11. Growth of HUVEC and HUVEC/PASMC spheroids in fibrin/plasma with varying bFGF and VEGF, at day 5, and labeling HUVEC with CellTracker Green. Fig. 12. Growth of HUVEC/PASMC spheroids in fibrin/plasma with VEGF alone, and labeling HUVEC with CellTracker Green.

Fig. 13. Growth of HUVEC/PASMC spheroids in fibrin/plasma with bFGF and VEGF 165, and labeling HUVEC with CellTracker Green.

Fig. 14. Effect of various agents on the growth of HUVEC/PASMC spheroids in fibrin/plasma with bFGF and VEGF 165, at day 4. Effect of transduced (A) Ad, CMV, ColVa2; (B) mATF, mAbrogen, hAbrogen, (C) hEndostatin, hAngiostatin, and hATF.

Detailed Description of the Invention

The invention relates to a multicellular in vitro composition that is capable of expressing, in vitro, quantifiable morphological changes due to angiogenic mechanisms occurring within the composition, such as, for example, endothelial cell lumenization and branching. The composition generally comprises (A) at least one spheroid of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix, and (C) a serum-free growth medium. The serum-free growth medium generally comprises sufficient nutrients and growth factors to allow growth of new vascular tissue when expression of quantifiable morphological changes due to angiogenesis in the sample is desired. The present invention generally comprises an in vitro composition comprising (A) at least one spheroid of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix; and (C) a serum-free growth medium.

The spheroids of the present invention comprise endothelial cells, and the endothelial cells may be derived from human tissue, such as, for example, human umbilical vein endothelial cells (HUVEC). Other endothelial cells may also be used where appropriate. The spheroids of the present invention also comprise smooth muscle cells, such as, for example, pulmonary artery smooth muscle cells (PASMC), or other smooth muscle cells may also be used where appropriate. The smooth muscle cells may be derived from human tissue.

In an embodiment of the present invention, the spheroids are brought into contact with a fibrin matrix. For example, a fibrin matrix may be prepared from fibrin precursors, such as fibrinogen and thrombin, which then form a fibrin matrix. In another embodiment, the composition further comprises at least one collagen gel, such as methylcellulose or matrigel.

The present inventive composition comprises a serum-free growth medium, such as EBM-2 or EGM-2. In general, the composition comprises a serum-free growth medium that allows the growth of the spheroid.

In one embodiment of the invention, the composition may comprise plasma.

Plasma is generally obtained, for example, by removing platelets from blood serum. Serum is generally obtained by removing red and white blood cells from whole blood. In another embodiment of the invention, the cells that comprise the spheroid have not been exposed to platelets, transforming growth factor, or platelet degranulation products.

The composition may further comprise at least one growth factor that promotes angiogenic growth of the spheroid. For example, the composition may comprise a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), or at least one of each. In one embodiment of the invention, the composition comprises bFGF and VEGF.

Generally, the ratio of endothelial cells to smooth muscle cells that are first combined to form a spheroid is a ratio that permits a spheroid to form and/or grow. In some embodiments the ratio of endothelial cells to smooth muscle cells ranges from 1 :2 to 10:1, such as, for example, 2:1 to 8:1. In one embodiment of the present invention, the ratio of endothelial cells to smooth muscle cells is about 4:1.

The composition may further comprise at least one agent that is known to promote or inhibit angiogenesis, or at least one agent whose effect on angiogenesis is unknown. In one embodiment of the invention, the composition further comprises at least one agent that is being screened for its ability to alter angiogenesis. Accordingly, the present invention also relates to a multicellular in vitro assay method for screening an agent for its ability to alter angiogenesis. The agent that is being screened may be chosen from known compounds, unknown compounds, natural compounds, synthetic compounds, compounds with a known biological activity, and compounds with no known biological activity. The agent being screened may be added in purified form or as part of a second added composition. Such second compositions may comprise, for example, an animal tissue extract, a plant tissue extract, a mineral extract, or combinations thereof. In one embodiment, the at least one agent that is being screened is a low molecular weight small molecule that can be synthesized in vitro. In another embodiment, the agent is being screened as a candidate for preventing or treating disorders or diseases in an animal involving undesired or abnormal angiogenesis. Disorders or diseases involving undesired or abnormal angiogenesis include, but are not limited to: tumor growth; tumor metastasis; diseases and conditions associated with chronic inflammation, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and lupus; diseases associated with corneal neovascularization; diseases associated with retinal/choroidal neovascularization; hemangiomas; Osler-Weber-Rendu disease; and hereditary hemorrhagic telangiectasia. Generally, the composition may be used in a multicellular in vitro assay for screening an agent for its ability to alter angiogenesis. In one embodiment, the assay method comprises: preparing an in vitro composition comprising (A) spheroids of endothelial cells and smooth muscle cells, (B) a fibrin matrix, and (C) a serum-free growth medium. The method may further comprise, preparing separate aliquots of the composition, wherein each aliquot comprises at least one spheroid; adding to a test aliquot an agent to be screened for its ability to alter angiogenesis; maintaining a control aliquot wherein the agent is not added; maintaining the aliquots for a sufficient time under sufficient conditions to allow a spheroid in at least one of the aliquots to express angiogenic features; measuring spheroid morphology in the aliquots at least once after angiogenic features are expressed; comparing the spheroid morphology measurements in the test aliquot to those of the control aliquot; determining the ability of the agent to alter angiogenesis in the spheroid; or combinations thereof. In one embodiment, control aliquots and test aliquots are each prepared in and analyzed in separate single well containers. In another embodiment, control aliquots and test aliquots are each prepared in and analyzed in the individual wells of a multi-well plate. In yet another embodiment, the control aliquot and/or test aliquot are prepared in and analyzed in a container with flow-through characteristics that would allow a continuous flow of nutrient medium and/or other soluble factors.

The spheroid of the present invention generally begins to exhibit angiogenic growth characteristics after the spheroid contacts the fibrin matrix.

In general, angiogenic growth characteristics may be observed within 7 days after the spheroids are contacted with the fibrin matrix. For example, angiogenic growth characteristics may be observed in 5-7 days, or 3-7 days. Angiogenic growth characteristics may include, but are not limited to, expression of cellular biochemistry (for example, expression of mRNA, peptides, glycoproteins, extracellular matrix components), or changes in cellular morphology (for example, endothelial cell lumenization structures, branching structures, or combinations thereof. In one embodiment, angiogenic growth characteristics of expression of cellular biochemistry is monitored and/or measured immediately before, during, and/or after the spheroid contacts the fibrin matrix , and subsequently optionally monitored for 5-7 days. In another embodiment, angiogenic growth characteristics of changes in cellular morphology is monitored and/or measured 5-7 days after the spheroid contacts the fibrin matrix. Expression of angiogenic characteristics of changes in cellular morphology includes, but is not limited to, endothelial cell lumenization structures, branching structures, or combinations thereof. Measurements of spheroid morphology may comprise, for example, identifying and recording the number or size of the endothelial cell lumenization structures, branching structures, or combinations thereof. Spheroid morphology structures may be measured in any physical dimension, such as, one dimension (e.g. width or length of all, or part, of a structure), two dimensions (e.g. the area of all, or part, of a structure), three dimensions (e.g. the volume of all, or part, of a structure), or combinations thereof. In one embodiment of the present invention, an automated image analysis process records and/or measures spheroid morphology. In another embodiment, an automated image analysis process records and/or measures spheroid morphology of multiple spheroid samples, wherein each individual spheroid sample is contained in its own single well of a multi-well plate.

In other embodiments of the present invention, the spheroids are labeled with at least one marker that can be used for measuring spheroid morphology. These markers may include, but are not limited to, chromogenic dyes, fluorescent labels, markers specific for endothelial cells, markers specific for smooth muscle cells, or combinations thereof. Fluorescent labels that may be used include, but are not limited to, CellTracker Green. In one embodiment of the present invention, the spheroids are labeled with at least one marker by labeling the endothelial cells, smooth muscle cells, or both before the spheroid is formed. Spheroid morphology may be measured by quantifying the fluorescence emission from labeled samples. Fluorescence emission may comprise, for example, fluorescence intensity, fluorescence wavelength spectrum, fluorescence lifetime, fluorescence quenching, fluorescence resonance energy transfer, or combinations thereof. Markers may be quantified, for example, with photomicroscopy, spectroscopy, or combinations thereof. In one embodiment, endothelial cells are labeled with a first marker and smooth muscle cells are optionally labeled with a second marker. For example, endothelial cells may be labeled with CellTracker Green.

Generally, the present inventive multicellular in vitro assay method for screening an agent for its ability to alter angiogenesis can be used for predicting in vivo outcomes for using the agent to prevent or treat disorders or diseases involving abnormal or excessive angiogenesis by referring to the measurements of the agent's ability to inhibit angiogenesis in the in vitro spheroid. In addition, the present inventive multicellular in vitro assay method can be used to screen a combination of at least two agents for their ability to alter angiogenesis in combination. In one embodiment, the method further comprises adding a second agent either before, after, or simultaneously with the addition of the first agent to a second test aliquot; measuring and comparing spheroid morphology between the control aliquot, the first test aliquot, and the second test aliquots; and determining the effect of the second agent on the activity of the first agent. Similarly, effects of three or more agents in combination can be determined by comparing the results of adding all the agents to a test aliquot, and having additional test aliquots wherein at least one of the agents being tested has been omitted.

Also, the compositions and methods disclosed herein are useful for studying and researching angiogenesis and the biological and chemical mechanisms related to angiogenesis. For example, the disclosed multicellular in vitro assay of angiogenesis is useful for studying the various complex processes involving extensive interplay between tissue types, cells, soluble factors, and extracellular matrix components and their contribution to normal angiogenic processes, abnormal angiogenic states, or the effects of specific pharmacological agents. The invention also relates to a kit comprising, in packaging of at least one part, an in vitro composition comprising (A) spheroids of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix; and (C) a serum-free growth medium, with sufficient nutrients to allow growth of new vascular tissue. The kit may be useful, for example, for analyzing the angiogenesis modulating effect of a known compound or unknown compound.

The invention also relates to a method of making a multicellular in vitro composition that is capable of expressing in vitro endothelial cell lumenization and braching, comprising combining (A) spheroids of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix; and (C) a serum-free growth medium, with sufficient nutrients and growth factors to allow growth of new vascular tissue. The present invention will be described in more detail with the aid of the examples which follow and which should be regarded as being illustrative and not limiting.

EXAMPLES

Example 1 : HUVEC spheroid formation and sprouting (Angiogenesis) assay.

1.1 HUVEC cell culture:

• HUVEC (Clonetics #CC-2519) was kept in liquid nitrogen. • The medium (Clonetics # CC-3162) contained EBM-2, was stored at 4"C, and supplement BulletKits, was stored at -20°C.

• Trypsin/EDTA (Gibco#25300-062), was stored at -20°C. PBS (Gibco P# 10010049), was stored at Room Temperature (RmT).

• Pipets (VWR#53283-700 1ml, 53283-706 5ml, 53283-708 10ml, 53283710 25ml) and Aspirate pipet (VWR #12777-014). Centrifuge tubes (VWR#21008-929 15ml, #21008-940 50ml).

• T150 flasks (VWR#29186-106) were pre-treated with 1% gelatin as indicate following.

• FBS (Hyclone #SH30071.03), DMSO (Sigma # D-2650). Freezing container (VWR# 55710-200), Cryo vial (VWR # 66008-706or 728).

Cryo box (VWR # 55709-968).

• Pipettors and tips (from Rainin). Bleach and biohazard bag for waste disposal.

1. EBM-2 was warmed to 37°C, and Bulletkits thawed. In sterile condition, all BulletKits components were added into the EBM-2 to make

HUVEC growth medium called EGM-2. The medium bottle was covered with foil and labeled with EGM-2 sticker. The EGM-2 was stored at 4°C, used within one month, and warmed up to 37°C whenever needed for cell culture. 2. In sterile condition, diluted 2% gelatin (Sigma #G1393) was stored at 4°C and warmed up to 37°C before using, to 1% with PBS. 5ml of 1% gelatin was added to each T150 flask. The flask was placed into a 37°C, 5% C0₂ incubator for 30 mins. The gelatin was aspirated out. The flask was then ready for use.

3. The cryovial of HUVEC was removed from liquid nitrogen and quickly dipped it into a 37°C water bath for 1-2 minutes until the contents thawed. The vial was immediately removed and wiped dry.

4. One T150 flask was labeled with cell type, passage number, strain number and date. In sterile condition, 25ml of EGM-2 was transfered into the T150 flask with pipets. A 1ml pipet was used to transfer HUVEC from cryovial to T150 flask for cell sending. The flask was placed into a 37°C, 5% C0₂ incubator and the flask flat was laid on its bottom overnight

(O/N).

5. The medium from the flask was aspirated, and replaced with 25ml of fresh EGM-2 medium, the day after seeding. The cells were examined every other day, the cells were fed with 10ml of EGM-2 every 3 days until they reached 70-80% confluence.

6. For subculturing the cells, the Trypsin/EDTA was removed from -20°C. After thawing, it was aliquoted into 40ml in sterile centrifuge tubes. At least one aliquot was refrozen at -20°C. At least one other aliquot was warmed up to room temperature and then used for subculturing the cells. 7. In a sterile field, the medium was aspirated from the T 150 cell flask, rinsed once with 10ml PBS and PBS was aspirated out. The cell were covered with 5ml Trypsin/EDTA. The cap of flask was closed. The cell layer was carefully examined microscopically until most of the cells were rounded up. The flask was rapped against the palm of the hand to release the majority of the cells from the culture surface.

8. After cells were released, the Trypsin was neutralized with 15 ml EGM-2. The cells were quickly transferred to a sterile 50 ml centrifuge tube. The cells were centrifuged at 220 x g for 5 minutes to harvest the cells. The supernatant was aspirated from the tube. The cells remained in the pellet. 9. In a sterile field, the cell pellet was resuspended in 5ml EGM-2 and mixed well. The cells were counted with a hemacytometer (VWR# 15 170-208). lOul of resuspended cells were mixed with lOul of Trypan Blue (Gibco# 15250-061). Then lOul of the mixture was placed into one of the two hemacytometer counting chambers. All of the unstained cells in each of the blue shaded corner squares were counted.

10. If the total cell count was n, then the cell suspension concentration was (n/2) x 10⁴ cells/ml. The total number of cells was (n/2) x 5 ml x 10⁴ Cells/ml. The amount needed was calculated. The cells were seeded at 8 x 10⁵ cells/T150 flask with 25-30 ml EGM2 with pre-treated and labeled flask. The flask was placed on its flat bottom in a 37°C, 5% C0₂ incubator. The cells were examined every other day, the medium was changed every 3 days until the cells reached 70-90% confluence. Then the cells were used or frozen as described below.

11. For subculture, step 6-9 were repeated. The total cells in a sample were calculated. The cells were centrifuged at 220 x g for 5 minutes to harvest the cells. The supernatant was aspirated from the tube. The cells were resuspended with FBS and 10% DMSO. The volume needed to make 1 x 10⁶ cells/ml suspension was calculated. 1ml was quickly aliquoted to each Cryo vial. The vial was sealed. Vials were placed in a room temperature freezing container with Isopropanol in the container (instructions on the freezing container were followed). The freezing container with vials was stored at -80°C for 24hrs. Then the vials were transferred to a Cryo box and the Cryo box was stored in liquid nitrogen.

1.2 HUVEC spheroid assay:

1. Pure powder of Methylcellulose (Sigma #M-0512, 4000 centipoises) (6g) was autoclaved with a magnetic stirrer in a 500ml capped bottle. The powder was dissolved in preheated 250ml EBM-2 (60°C) and stirred for 20min. Another 250ml EBM-2, at room temperature, was added to a final volume of 500ml and this stock solution was stirred for l-2h at 4°C. Then the solution was cleared by centrifiigation (5000g x 2hr, room temperature). The resulting solution, called methocel stock, was collected.

All processes were under sterile conditions. 2. The HUVEC cells were grown in EGM-2 and trypsinized and counted as indicated above. A defined number of cells (750cells in 150ul medium/ well of 96 well plate) were mixed with methocel containing medium which was 20% methocel stock solution and 80% EGM-2. For example, for one 96-well-plate, 7.5 x 10⁴ cells were suspended in 15ml of well mixed medium, which contained 12ml of EGM-2 and 3ml of methocel stock. 150ul of the cells were distributed to each well of the 96-wellplate (Cat, # 650185 from Greiner, suspension culture, U-form). After 24h at 37°C in the incubator, all suspended cells in each well contributed to the formation of a single endothelial cell spheroid, called standard sized spheroid.

3. The following reagents were prepared:

Fibrinogen stock: 4 mg/ml Fibrinogen (Sigma #F-4753) was prepared in EBM2, filtered with 0-2um filter (VWR#28144-040) under sterile conditions. The fibrinogen stock was prepared fresh for each assay. Thrombin stock: lOOU/ml thrombin (Sigma #T-4648) was prepared in

EBM-2, filtered and aliquoted to small volume. The thrombin stock was stored at -20°C, and thawed before use.

Plasma stock: lyophilized plasma (Sigma #P4639) was dissolved as the indicated volume with ddH₂0, filtered and aliquoted to small volumes. The plasma stock was stored at -20°C, and thawed before use.

2x assay control medium: 2 sets of BulletKits were added into one EBM-2, but the FBS, bFGF and VEGF Bullets from the kits were not added to the EBM-2 medium. So the 2x assay control medium was the 2x EGM-2 without any FBS, bFGF and VEGF. 2x assay medium: 2x assay control medium was combined with l-2ug/ml bFGF ('R&D#234FSE) and l-2ug/ml VEGF (R&D #293-VE).

4. 0.25ml of 2x assay or control medium was mixed with 0.25ml of Fibrinogen stock, lOul of Thrombin stock, and lOul of Plasma stock. The mixture was quickly spread on the bottom of one well of a 24-well tissue culture plate (VWR # 29442-044). The culture plate was kept at 37°C in an incubator until the fibrin gels solidified. 5. The spheroids were harvested within 24h, using standard pipets (VWR # 14670-114). 12 spheroids were transferred into one 15ml centrifuge tube with 10ml of PBS, and centrifuge at 300-500 xg, for 3 minutes. The supernatant was removed and the tube was shortly scratched over a rough underground to loosen the pellet. The loosened pellet was then not allowed to stay in the tube for more than 15-30 minutes, otherwise the spheroids comprising the pellet would have stuck together.

6. Each pellet was overlaid (not mixed) with 0.25 ml of 2x assay or control medium, lOul of Plasma stock, lOul of Thrombin stock ,and 0.25ml of Fibrinogen stock in each 15ml spheroid-containing tube. The contents of the tube were mixed well and quickly spread onto the corresponding fibrin gel of each well of a 24-well-plate. The plates were incubated at 37°C until the gels solidified. 500 ul of IX corresponding medium was added on top of the gel. The plates were placed back in incubator. 7. Results were measured around 5-7 days. Angiogenic growth was observed and recorded by using photomicroscopy.

8. For co-culture of HUVEC with PASMC (Cloneties #CC-2581, # CC-3182 for medium), spheroids were set up as 800 cells of HUVEC + 200 cells of PASMC/spheroid as 4:1. 9. Where a label was needed, the cells were labeled following

Sigma# PKH67-GL protocol before cells were combined to form spheroids.

Claims

1. An in vitro composition comprising (A) at least one spheroid of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix; and (C) a serum-free growth medium.

2. The composition according to claim 1, further comprising plasma.

3. The composition according to claim 1, wherein the spheroid has not been exposed to platelets or transforming growth factor.

4. The composition according to claim 1, wherein the spheroid has not been exposed to platelet degranulation products.

5. The composition according to claim 1, wherein the endothelial cells are selected from human umbilical vein endothelial cells (HUVEC).

6. The composition according to claim 1, wherein the endothelial cells are derived from human tissue.

7. The composition according to claim 1, wherein the smooth muscle cells are selected from pulmonary artery smooth muscle cells

(PASMC).

8. The composition according to claim 1, wherein the smooth muscle cells are derived from human tissue.

9. The composition according to claim 1, wherein the fibrin precursors are fibrinogen and thrombin.

10. The composition according to claim 1, wherein the spheroid comprises endothelial cell lumenization structures, branching structures, or combinations thereof.

11. The composition according to claim 1, further comprising bFGF, VEGF, or both.

12. The composition according to claim 1, further comprising at least one agent that is known to promote or inhibit angiogenesis.

13. The composition according to claim 1, further comprising at least one agent that is being screened for its ability to alter angiogenesis.

14. The composition according to claim 13, wherein the agent that is being screened is a low molecular weight small molecule that can be synthesized in vitro.

15. The composition according to claim 13, wherein the agent that is being screened can be administered to an animal to prevent or treat disorders or diseases involving abnormal angiogenesis.

16. The composition according to claim 1, wherein the ratio of endothelial cells to smooth muscle cells is a ratio that allows angiogenic growth of the spheroid.

17. The composition according to claim 11, wherein the ratio of endothelial cells to smooth muscle cells ranges from 1:2 to 10:1.

18. The composition according to claim 1, wherein the ratio of endothelial cells to smooth muscle cells is about 4:1.

19. The composition according to claim 1, wherein the serum-free growth medium is selected from EBM-2 or EGM-2.

20. The composition according to claim 1, further comprising at least one collagen gel.

21. The composition according to claim 20, wherein the collagen gel is selected from methylcellulose or matrigel.

22. A multicellular in vitro assay method for screening an agent for its ability to alter angiogenesis comprising: preparing an in vitro composition comprising (A) spheroids of endothelial cells and smooth muscle cells, (B) a fibrin matrix, and (C) a serum-free growth medium; preparing separate aliquots of the composition, wherein each aliquot comprises at least one spheroid; adding to a test aliquot an agent to be screened for its ability to alter angiogenesis; maintaining a control aliquot wherein the agent is not added; maintaining the aliquots for a sufficient time under sufficient conditions to allow a spheroid in at least one of the aliquots to express angiogenic features; measuring spheroid morphology in the aliquots at least once after angiogenic features are expressed; comparing the spheroid morphology measurements in the test aliquot to those of the control aliquot; and determining the ability of the agent to alter angiogenesis in the spheroid.

23. The method according to claim 22, wherein the composition further comprises at least one FGF, at least one VEGF, or combinations thereof.

24. The method according to claim 22, wherein the endothelial cells are selected from human umbilical vein endothelial cells (HUVEC).

25. The method according to claim 22, wherein the smooth muscle cells are selected from PA smooth muscle cells (PASMC).

26. The method according to claim 22, wherein the spheroid is prepared before the spheroid is combined with the fibrin matrix.

27. The method according to claim 22, wherein the spheroid is combined with the fibrin matrix by co-mixing the spheroid with fibrin matrix precursors.

28. The method according to claim 27, wherein the fibrin matrix precursors comprise thrombin and fibrinogen.

29. The method according to claim 22, wherein the initial ratio of endothelial cells to smooth muscle cells in the spheroid is about 4:1.

30. The method according to claim 22, wherein spheroid morphology comprises endothelial cell lumenization structures, branching structures, or combinations thereof.

31. The method according to claim 22, wherein measuring spheroid morphology comprises identifying and recording the number or size of the endothelial cell lumenization structures, branching structures, or combinations thereof.

32. The method according to claim 22, wherein the spheroid morphology is measured in one dimension, two dimensions, three dimensions, or combinations thereof.

33. The method according to claim 22, wherein the spheroid morphology is measured 5-7 days after the spheroids are combined with the fibrin matrix.

34. The method according to claim 22, further comprising labeling the spheroids with at least one marker that can be used for identifying or measuring spheroid morphology.

35. The method according to claim 34, wherein the spheroids are labeled with the marker by labeling the endothelial cells or smooth muscle cells before the spheroid is formed.

36. The method according to claim 22, wherein the agent that is being screened is a low molecular weight small molecule that can be synthesized in vitro.

37. The method according to claim 22, wherein the agent that is being screened can be administered to an animal to prevent or treat disorders or diseases involving abnormal angiogenesis.

38. The method according to claim 22, further comprising predicting in vivo outcomes for using the agent to prevent or treat disorders or diseases involving excessive angiogenesis by referring to the measurements of the agent's ability to inhibit angiogenesis in the in vitro spheroid.

39. The method according to claim 22, wherein the agent that is being screened is added to the test aliquot as a single dose or in multiple doses.

40. The method according to claim 22, further comprising adding a second agent either before, after, or simultaneously with the addition of the first agent to a second test aliquot; measuring and comparing spheroid morphology between the aliquots; and determining the effect of the second agent on the activity of the first agent.

41. A kit for analyzing the angiogenesis modulating effect of a compound, comprising an in vitro composition comprising (A) spheroids of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix; and (C) a serum-free growth medium, with sufficient nutrients to allow growth of new vascular tissue.

42. A method of making a multicellular in vitro composition that is capable of expressing in vitro endothelial cell lumenization and braching, comprising combining (A) spheroids of endothelial cells and smooth muscle cells, (B) a fibrin matrix or precursors for a fibrin matrix; and (C) a serum-free growth medium, with sufficient nutrients and growth factors to allow growth of new vascular tissue.