WO2011150375A2

WO2011150375A2 - Endothelial colony forming cell culture medium

Info

Publication number: WO2011150375A2
Application number: PCT/US2011/038414
Authority: WO
Inventors: Mervin C. Yoder; Lan Huang
Original assignee: Indiana University Research And Technology Corporation
Priority date: 2010-05-28
Filing date: 2011-05-27
Publication date: 2011-12-01
Also published as: WO2011150375A3

Abstract

Disclosed herein is cell culture media comprising umbilical cord plasma that is substantially free of serum. The media may be supplemented with one or more growth factors, hrbFGF, hrEGF, hrVEGF₁₆₅, hrVEGF₁₂₁, hrSCF, SDFlα, hrIL-6, and any combination thereof. The media may be used to expand cells in culture, such as endothelial colony forming cells, prior to implantation into a patient.

Description

ENDOTHELIAL COLONY FORMING CELL CULTURE MEDIUM

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No. 61/349,599, filed on May 28, 2010, the entire disclosure of which is incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates generally to cell culture. More specifically, the present disclosure relates to substantially serum-free culture media for endothelial cell colony forming cells.

BACKGROUND AND SUMMARY

The progenitor cells for the endothelial lineage play critical roles in vascular homeostasis and regeneration in adult subjects. High proliferative potential endothelial colony forming cells (HPP-ECFCs) have been identified as endothelial progenitor cells (EPCs) with robust proliferative potential in vitro and vessel-forming ability in vivo. Additionally, recent studies have revealed that the concentration of ECFCs in circulation increases after vascular ischemia, which implies a possible contribution to vascular repair. Thus, ECFCs may be useful for vascular regenerative therapies. However, the therapeutic use of ECFCs is hampered by challenges in culturing the cells.

Current protocols for in vitro expansion of ECFCs mostly depend on the presence of fetal bovine serum or fetal calf serum (FBS or FCS) in the culture medium. Importantly, FBS/FCS may contain potentially harmful xenogenic compounds associated with risks of transmitting infectious agents, such as prions, or inducing immune reactions when used in a transplantation setting. Of particular concern is the potential for transmission of deadly diseases such as spongiform encephalopathies. Presently, there are no serum free media suitable for the expansion of ECFCs. Therefore, a defined animal serum-free culture medium for in vitro isolation and expansion of ECFCs is needed.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows (A, B) isolated human cord blood (HCB) endothelial colony forming cell-derived endothelial cell colonies from umbilical cord blood (UCB) mononuclear cells (MNCs) by using serum reduced medium (SRM). Figure 2 shows the quantitation of the clonogenic and proliferative potential of single ECs derived from HCB cultured in SRM.

Figure 3 shows the potential of HCB-derived ECFC cultured in SRM to form functional microvessels in immunodeficient mice.

Figure 4 shows the size distribution of the microvessels formed by human-cord- blood ECFCs.

Figure 5 shows the genomic stability in HCB ECFCs cultured in SRM.

DETAILED DESCRIPTION

While the invention is susceptible to various modifications and alternative forms, specific embodiments will herein be described in detail. It should be

understood, however, that there is no intent to limit the invention to the particular forms described, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the invention.

Disclosed herein is cell culture media substantially free of serum. The media may be used for the expansion of transplantable cells, such as ECFCs. As used herein, the phrases "media substantially free of serum" or "serum free media" or "substantially serum free media" or "serum reduced media (SRM)" relate to cell culture media that contains no serum other than in trace or contaminant amounts. Serum reduced media (SRM) or media substantially free of serum is understood by those of skill in the art to refer to a cell culture media that contains no added serum derived from an animal, such as fetal bovine serum or fetal calf serum.

In one embodiment, a substantially serum free media comprising human umbilical cord plasma is disclosed. In one embodiment, a substantially serum free media comprising human umbilical cord plasma and at least one growth factor is disclosed. In one embodiment, a substantially serum free media comprising human umbilical cord plasma and at least one growth factor is disclosed, wherein the at least one growth factor is selected from the group consisting of a human epidermal grown factor (hrEGF), a human recombinant vascular endothelial growth factor 165 (hrVEGF165), a human recombinant vascular endothelial growth factor 121 (hrVEGFm), a human recombinant stem cell factor (hrSCF), a stromal cell derived 1 alpha (SDF la), and a human recombinant interleukin 6 (hrIL-6), and any combination thereof. In various embodiments, the human umbilical cord plasma is present at a level of about .5% to about 3%, or from about .5% to about 2.5%, or about .5% to about 1%, or about .5% to about 1.5%, or from about 1% to about 3%, or about 1% to about 2%, about 1.5% to about 3%, or about 2% to about 3%, v/v. These various concentrations of umbilical cord plasma are contemplated where the term "about" is not included.

In various embodiments, each individual growth factor in the substantially serum free media may be present in a serum free media at a level of about 5ng/ml to about 30 ng/ml, about 5 ng/ml to about 25 ng/ml, or about 5 ng/ml to about 10 ng/ml, or about 10 ng/ml to about 25 ng/ml, or about 15ng/ml to about 30 ng/ml, or about 20 ng/ml to about 30 ng/ml. These various concentrations of growth factors are also contemplated where the term "about" is not included.

In one embodiment, the substantially serum free media further comprises at least one antibiotic. Illustrative antibiotics include, but are not limited to, penicillin, streptomycin, and the like. In one embodiment, the substantially serum free media further comprises a basal medium.

The media may be used to expand cells in culture. As used herein, "expanding" isolated human endothelial colony forming cell means increasing the number of individual cells in culture as compared to an initial starting number of cells. In one embodiment, a method of expanding the number of human endothelial colony forming cells (ECFC) in culture is disclosed, the method comprising culturing the ECFCs in a substantially serum free media as described herein.

In one embodiment, a composition for implantation into a patient is disclosed, the composition comprises ECFCs and a substantially serum free media.

ECFCs may be isolated from cord blood and/or vessel walls, and may have one or more of the following characteristics: (a) expression of cell surface antigens that are characteristic of endothelial cells, such as CD31, CD105, CD146, and CD144; (b) no expression of cell surface antigens that are characteristic of hematopoietic cells, such as CD45 and CD14; (c) ingestion of acetylated LDL; and (d) formation of capillary-like tubes in extracellular matrix proteins, such as Matrigel™.

In one illustrative aspect, expanded human endothelial colony forming cells express a surface marker phenotype comprising one or more of CD31, CD34, CD144, CD146, VEGFR1, VEGFR2, and VEGFR3 and do not express one or more of CD1 lb, CD14, CD45, and AC 133. In one embodiment, a method of treating a patient is disclosed, the method comprising the steps of expanding ECFCs in a substantially serum free medium and

administering the ECFCs to a patient. The patient may be any mammal, such as a human. In one embodiment the ECFCs are harvested from the same patient that is to be administered the expanded ECFCs.

In one embodiment, the expanded ECFCs may be administered to the patient by traditional routes, for example, intravenous injection, intramuscular injection,

subcutaneous injection, retrograde venous injection, arterial injection, and surgical implantation.

In one embodiment, a method of forming vessels in vivo is disclosed, the method comprising the step of implanting a composition in an animal, the composition comprising endothelial colony forming cells in media substantially free of serum, wherein the cell forms at least one vessel in the animal.

In one embodiment, ECFCs expanded in substantially serum free media may be cultured within a support material prior to administration to a patient. A "support material" as used herein refers to any biologically compatible substance that can support the association of ECFCs to form blood vessels. Suitable support materials include, but are not limited to biologically compatible polymer material such as collagen, elastin, fibrinogen, fibrin, fibronectin, gelatin, laminin, vitronectin, hyaluronan, heparan sulfate, agar, agarose, alginate, chitosan, collagen-fibronectin, collagen-gelatin, collagen-agarose, collagenchitosan, collagen-chitosan- agarose, collagen-chitosan-gelatin, collagen-vitronectin-agarose, collagen-vitronectin-gelatin, collagen- vitronectin-chitosan collagen- fibronectin-agarose, collagen-fibronectin-gelatin collagen- fibronectin-chitosan, collagen-laminin-agarose, collagen-laminin-gelatin, collagen- laminin-chitosan, and combinations thereof. The support material may further incorporate additional agents such as excipients, growth factors, vitamins, minerals, ions, gases, crosslinking agents, active agents, carriers, and combinations thereof.

EXAMPLES

Human Endothelial Serum Free Medium (SFM; Invitrogen, Grand Island, NY) was supplemented with 20 ng/ml human recombinant basic fibroblast growth factor (hrbFGF) (Invitrogen), 10 ng/ml human recombinant epidermal growth factor (hrEGF) (R&D, Minneapolis, MN), 10 ng/ml human recombinant vascular endothelial growth factor 165 (hrVEGF165) (R&D), 10 ng/ml human recombinant VEGF121

(hrVEGF121) (R&D), 10 ng/ml human recombinant stem cell factor (hrSCF) (R&D), 5 ng/ml stromal cell derived 1 alpha (SDFla) (R&D), 10 ng/ml human recombinant interleukin 6 (hrIL6) (R&D), 1.5% human umbilical cord plasma (HCP) and 1.0%

penicillin/streptomyocin (Invitrogen), were mixed to create an exemplary serum reduced medium (SRM) of the present disclosure.

Human recombinant basic fibroblast growth factor (hrbFGF), human recombinant epidermal growth factor (hrEGF), human recombinant vascular endothelial growth factor 165 (hrVEGFi6s), human recombinant VEGF 121 (hrVEGFm), human recombinant stem cell factor (hrSCF), stromal cell derived 1 alpha (SDFla), human recombinant interleukin 6 (hrIL6) were added to the media both individually and in

combination to examine their ability to promote ECFC emergence and expansion from human MNCs.

As a control, human EGM-2 medium (Lonza, Walkersville, MD) was supplemented with 10% FBS (Hyclone, Logan, UT) and 1.0% penicillin/streptomycin, and called complete EGM-2 medium, or cEGM-2. While human umbilical cord plasma substituted for 10% FBS, it was found that ECFCs were unable to be isolated and expanded in the absence of the cord plasma.

Preparation of pooled HCP

Human umbilical cord blood (UCB) samples (50-100 mL) were collected in heparin-coated syringes (20 to 30 USP units of heparin/mL of blood) from healthy newborns (38-40 weeks gestation). UCB was diluted 1:1 with Dulbecco's Phosphate Buffered Saline (DPBS) (Invitrogen) and overlaid onto Ficoll-Paque PLUS (GE Healthcare, Piscataway, NJ) according to the manufacturer's instructions. Cells were centrifuged for 30 minutes at room temperature at 1500 rpm. After centrifugation, the MNCs were collected for culturing endothelial cell colonies, and the supernatant was collected for preparing human cord plasma (HCP). Subsequently, the supernatant was aliquoted and frozen at -80°C. After thawing, aliquots with the same volume from at least 20 samples were pooled and sterilely filtered through a 0.2 μιη filter. The pooled HCP was then added to the media.

Isolation and culture of UCB-derived ECFCs

MNCs were isolated and washed with DPBS. For outgrowth of ECFC colonies, MNCs either were resuspended in SRM or cEGM-2 medium. MNCs (3 x 10⁷ /well) were seeded onto 6-well tissue culture plates pre-coated with Type I rat-tail collagen (BD Biosciences; Bedford, MA) and cultured as previously described (Ingram DA 2004). Spindle- shaped ECFC colonies emerged sequentially from the MNCs and the first day of ECFC colony emergence was recorded. The frequency of ECFC colonies was determined by measuring the total number of colonies in the primary culture on day 10 (as no ECFC ever emerged at a later time point). Subsequently, the ECFC-derived ECs were released from the primary culture dish by TrypLETM Express (Gibco, Grand Island, NY) and replated onto 25 cm² tissue culture flasks pre-coated with Type I rat-tail collagen for subsequent passage.

Immunophenotyping of ECFC derived ECs

Early passaged (1-2) ECFC-derived ECs (5 x 10⁴) were incubated at 4°C for 30 minutes in 100 ill of medium with varying concentrations of the primary or isotype control antibody as outlined below, washed three times, and analyzed by fluorescence-activated cell sorting (FACS^®) (Becton Dickinson, San Diego, CA). The primary antibodies used included antihuman CD31 conjugated to phycoerythrin (PE) (BD Biosciences Pharmingen; Bedford, MA), anti-human CD34 conjugated to allophycocyanin (APC) (BD Biosciences Pharmigen), antihuman CD 144 conjugated to PE (BD Biosciences Pharmingen), anti -human CD 146 conjugated to PE (BD Biosciences Pharmingen), anti-human cKIT conjugated to APC (eBioscience; San Diego, CA), anti-human VEGFRl conjugated to PE (BD Biosciences

Pharmingen), anti-human VEGFR2 conjugated to fluorescein isothiocyanate (FITC) (BD Biosciences Pharmingen), antihuman VEGFR3 conjugated to APC (R&D), anti-human Nrp 1 conjugated to PE (Miltenyi Biotec; Auburn, CA), anti-human Nrp2 (R&D) conjugated to Alexa Fluor 647 (Molecular Probes, Eugene, OR), anti-human CD14 conjugated to PE (BD Biosciences Pharmingen), antihuman CD45 conjugated to FITC (BD Biosciences Pharmingen), anti-human AC- 133 conjugated to APC (Miltenyi Biotec) and anti- human CXCR4 conjugated to FITC (BD Biosciences Pharmingen). For negative controls, directly conjugated mouse IgG isotypes (BD Biosciences Pharmingen) were used.

Single cell clonogenic assays

Early passaged (1-2) ECFC-derived ECs were plated at one cell per well into 96 well plates pre-coated with Type I rat-tail collagen in 200 pi of cEGM-2 medium. Cells were cultured at 37°C in a humidified incubator with 5% CO2. Media were changed every five days. After 14 days of culturing, cells were fixed with 4% paraformaldehyde (Sigma; St. Louis, MO) in phosphate-buffered saline for 30 minutes at room temperature, then washed twice, stained with 1.5 pg/ml DAPI, and examined for the growth of ECs. Those wells containing two or more cells were identified as positive for proliferation under a fluorescent microscope at lOx magnification. Wells containing fewer than 50 cells were counted by visual inspection with a fluorescent microscope at 40x magnification. For those wells with more than 50 cells, colonies were imaged and cell number quantified using an Image J1.36v program (Wayne Rasband, NIH).

In vivo matrix implantation assays

Early passaged (3-5) ECFC-derived ECs (2 x 10⁶ cells/mL) were suspended in a 1.5 mg/mL collagen-fibronectin matrix as previously described (Yoder 2007, Critser 2010). Aliquots (250pl) were pipetted into wells of 48 well plates, allowed to polymerize at 37°C for 30 minutes, and covered with 500pl of culture medium for overnight incubation at 37°C, in 5% CO₂. After 18 hours of ex vivo culture, cellularized matrices were implanted into the flanks of 6- to 8-week-old NOD/SCID mice. After 14 days, mice were euthanized and the grafts were harvested, fixed in formalin-free zinc fixative (BD Biosciences), paraffin embedded, bisected, and sectioned (6 μιη) for analysis by histology and

immunohistochemistry (N=6).

Histology and Immunohistochemistry

Sections were stained and paraffin-embedded tissue sections were deparaffinized and then either directly stained with hematoxylin and eosin (H&E) or immersed in retrieval solution (Dako, Carpenteria, CA) for 20 minutes at 90-99°C. Slides were incubated at room temperature for 30 minutes with anti-human CD31 (clone JC70/A, Abeam), followed by a 10 minute incubation with LASB2 link-biotin and streptavidin-HRP (Dako), then developed with DAB (Vector, Burlingame, CA) solution for 5 minutes.

Statistical Analysis

Results are shown as the mean ± the standard error of the mean (SEM). Data were analyzed with ANOVA; parametric test and significant differences were set at P < 0.05. All analyses were performed using GraphPad InStat software (GraphPad Software Inc, La Jolla, CA). Isolation and expansion of human UCB ECFCs

Human ECFC-derived EC colonies have been shown to be capable of isolation from low-density MNCs in umbilical cord blood by utilizing cEGM-2 medium. To evaluate whether SRM is able to promote ECFC outgrowth and proliferation, two culture media were directly compared. MNCs from the same donor were divided into portions with half of them cultured in SRM and the rest in cEGM-2. The first ECFC colonies were detected after 4.35 ± 0.25 days in SRM, compared to 6.10 ± 0.38 days in cEGM-2 (p < 0.001, Figure 1A) (Time of initial ECFC derived EC colonies emerged from MNCs after culture initiation in SRM and cEGM-2. Results represent the mean number of days before initial EC appearance ± SEM (n = 23, *P < 0.001)). No difference was observed in the frequency of ECFCs recovered on day 10 under these two conditions (Figure IB) (Number of ECFC-derived EC colonies outgrown per 10⁷ MNCs after 10 days of culture initiation in SRM and cEGM-2. Results represent the average number of EC colonies ± SEM (n = 23)). Colonies in SRM displayed a cobblestone appearance with variations in colony size, which indicated their

heterogeneous proliferative abilities (Figure 1C). Representative photomicrographs of individual human ECFC-derived EC colonies from UCB in SRM. Scale bar represents 100 μη.

Phenotypic characterization of human UCB ECFCs

The ECFC colonies expanded and formed an endothelial monolayer in both types of culture media conditions. Immunophenotyping of the endothelial monolayer (Figure 2) revealed that ECs cultured in SRM expressed endothelial cell-surface antigens CD31, CD34, CD144, CD146, VEGFR1, VEGFR2 and VEGFR3, which was similar with those cultured in cEGM-2. Immunophenotyping of EC from the cultured monolayer derived from human cord blood ECFC in SRM (A) and cEGM-2 (B) by fluorescence cytometry. Similar to cells grown in cEGM-2, the ECs cultured in SRM expressed CD31, CD34, CD 144, CD 146, Fltl, Flkl, Flt4, andNrp2 but not CD45, CD14, CDl lb or AC133. However, the expression of cKIT (the receptor of stem cell factor, SCF) and CXCR4 (the receptor of stromal cell derived factor 1 alpha, SDF 1 a) was higher in ECs in SRM than in cEGM-2. Further, the EC colonies cultured in SRM did not express the hematopoietic cell surface antigens CDl lb, CD14, CD45 or AC133, which indicates that the HCP-supplemented culture environment was devoid of hematopoietic cell contamination.

Clonogenic ability maintained in human UCB ECFCs A complete hierarchy of ECFC in human peripheral blood and UCB derived ECs, based on proliferative and clonogenic ability has been previously described. To determine whether such a proliferative hierarchy is also present in the ECs cultured in SRM, a single-cell clonogenic assay was performed. After single cells were plated in culture, some cells didn't divide, while other cells divided and formed colonies of different sizes comprised of varying cell numbers. The frequency of single cells undergoing division was similar between samples cultured in SRM and those from cEGM-2 (28.10 ± 21.04 vs 34.30 ± 20.89, respectively). Moreover, the entire hierarchy of ECFCs, composed of high proliferative (HPP)-, low proliferative (LPP)-ECFC, endothelial-cluster and non-dividing mature ECs, was exhibited in ECs cultured in SRM (Figure 2). Figure 2 shows the distribution of colony sizes, where colonies were derived from single ECs grown in individual wells after 14 days of culture. The complete hierarchy of ECFCs was present in ECs cultured in SRM; that is similar to those grown in cEGM-2. Inset chart is the percentage of single ECs dividing at least once after growing 14 days in culture. No statistical difference in this frequency was observed between cells grown in SRM and those in cEGM-2. (N = 5).

In vivo formation of chimera blood vessels

Human UCB-derived ECFCs have been demonstrated to possess the potential to form de novo blood vessels when suspended in a collagen-fibronectin matrix or Matrigel and implanted subcutaneously into immunodeficient mice [Au P 2007, Melero-martin 2008]. To test the in vivo vessel-forming ability of UCB ECFCs cultured in SRM, the same methods were employed. After 14 days of carrying implants containing ECFC cultured in SRM or cEGM2, the mice were euthanized, the grafts were harvested, and analyzed for human or murine blood vessel formation.

H&E staining revealed the formation of human microvessels perfused with murine red blood cells in the graft, indicating human vessel anastomoses with the

surrounding murine vasculature. H&E staining indicates microvessel formation in collagen- fibronectin gel after 14 days of implantation in NOD/SCID mice. Anti-human CD31 staining further confirms the human origin of these vessels. To further verify the human origin of these vessels, an immunohistochemistry study with a specific anti-human CD31 antibody was conducted. Thus, ECFC progeny cultured in SRM can also form functional human-murine chimeric vessels in a short-term xenograft model of blood vessel formation similar to cEGM2 media cultured cells. Quantification of human microvessels that carry murine erythrocytes (Figure 3) showed that there was no statistical difference between the implanted cells cultured in these two culture media (SRM vs CEGM-2 is 28.48 + 14.86 vs. 14.73 + 6.69 vessels/mm², the number of vessels formed by human cord-blood-derived ECFCs and perfused with murine red blood cells per mm² in the gel after 14 days of implantation. (n=6)). Furthermore, the size distribution of these functional microvessels formed by ECFC cultured in SRM was similar to those cultured in cEGM-2 (Figure 4). The size distribution of the microvessels formed by human-cord-blood ECFCs. These data indicate that there is no difference in the vessel-forming abilities of ECs cultured in SRM versus those in cEGM-2.

Genetic Stability

In an examination of the genetic stability of ECFC cultured in SRM as compared to cEGM-2, there was no significant difference in the incident of polyploid and aneuploid formation during 30 days of tissue culture. Fluorescence in situ hybridization (FISH) analysis of ECs using centromere probes specific for the X chromosome and chromosome 17. Nuclei are stained with DAPI. Normal (diploid) female cells display two X chromosomes and two chromosome 17s. Tetraploid female cells display four X chromosomes and four chromosome 17s. As shown in Figure 5, FISH analysis of > 200 ECs after 30 days of culture initiation revealed a similar frequency of diploid content in cells grown in SRM and in cEGM-2.

Chromosomally aberrant cells (tetraploid and aneuploid) were detectable at a low incidence in both culture conditions. N=4.

Claims

WHAT IS CLAIMED IS

1. Cell culture media comprising umbilical cord plasma, wherein the media is substantially free of serum.

2. The media according to claim 1 further comprising at least one growth factor.

3. The media according to claim 2 wherein the at least one growth factor is selected from the group consisting of human recombinant basic fibroblast growth factor (hrbFGF), human recombinant epidermal grown factor (hrEGF), human recombinant vascular endothelial growth factor 165 (hrVEGFi6₅), human recombinant vascular endothelial growth factor 121 (hrVEGFm), human recombinant stem cell factor (hrSCF), stromal cell derived 1 alpha (SDFla), human recombinant interleukin 6 (hrIL-6), and any combination thereof.

4. The media according to claim 1 further comprising an antibiotic.

5. Cell culture media substantially free of serum, the media comprising human umbilical cord plasma, human recombinant basic fibroblast growth factor (hrbFGF), human recombinant epidermal grown factor (hrEGF), human recombinant vascular endothelial growth factor 165 (hrVEGFi6₅), human recombinant vascular endothelial growth factor 121 (hrVEGFi2i), human recombinant stem cell factor (hrSCF), stromal cell derived 1 alpha (SDFla), and human recombinant interleukin 6 (hrIL-6).

6. The cell culture media of claim 5 further comprising an antibiotic.

7. A method of expanding the number of human endothelial colony forming cells (ECFC) in culture comprising the step of contacting the ECFCs with a media according to any of claims 1 to 6.

8. The method of claim 7, wherein the ECFCs express one or more cell surface antigens selected from the group consisting of CD31, CD34, CD 144, CD 146, Fltl, Flkl, Flt4, andNrp2.

9. The method of claim 7, wherein the ECFCs do not express cell surface antigens CDl lb, CD14, CD45 or AC133.

10. A composition for implantation into a patient, the composition comprising ECFCs and a substantially serum free media according to any of claims 1 to 6.

11. A method of forming vessels in vivo comprising the step of implanting a composition in an animal, the composition comprising endothelial colony forming cells in media substantially free of serum according to any of claims 1 to 6, wherein the cell forms at least one vessel in the animal.