WO2009021333A1

WO2009021333A1 - Blood vessel extraction from umbilical cord

Info

Publication number: WO2009021333A1
Application number: PCT/CA2008/001466
Authority: WO
Inventors: Jane Elizabeth Ennis; Elaine Cheng; Jeffery Donald Turner
Original assignee: Tissue Regeneration Therapeutics Inc.
Priority date: 2007-08-14
Filing date: 2008-08-13
Publication date: 2009-02-19

Abstract

The invention provides a method for processing umbilical cord that provides exposed intact cord vasculature for subsequent processing. The method includes the steps of : anchoring an excised umbilical cord at a location along a length thereof; cutting the cord at a site proximal to the anchored location to penetrate an epithelium while leaving internal vasculature intact; and removing the epithelium by peeling the epithelium from the cut, along the length of the cord, and away from the anchored location.

Description

BLOOD VESSEL EXTRACTION FROM UMBILICAL CORD

FIELD OF INVENTION

This invention relates to progenitor cells that are extractable from the perivascular region of blood vessels including veins and arteries resident in the umbilical cord. More particularly, the invention relates to new methods and procedures that facilitate the harvesting of such cells and source tissue.

BACKGROUND OF THE INVENTION

The external surface of the vein and two arteries that comprise the umbilical cord vasculature has proven to be a fertile source of valuable progenitor cells. Indeed, the collection and preservation of these cells is now commercially available to parents wishing to retain them for future purposes. It has been shown that progenitor cells extracted from this region comprise cells that can differentiate into various mesenchymal cell types including osteoblasts, chondrocytes, and adipocytes, as well as myocytes. A certain fraction of the extracted progenitor cell population also is negative for both the MHC-I and MHC-II phenotypes.

Suitable methods of extracting and isolating these progenitor cells are disclosed in a variety of publications including US2005/0148074 published July 7, 2005 and by Sarugaser et al in Stem Cells, 23, 220-229 (2005), the entire disclosures of which are incorporated herein by reference. To isolate progenitor cells from umbilical cord, these publications describe a technique whereby the excised cord is first sectioned transversely to provide workable segments that are then cut longitudinally, to permit removal of the associated amniotic epithelium, and to expose the underlying vasculature bearing a coating of connective tissue known as Wharton's jelly, which is a matrix comprised principally of a collagen and ground substance. The treated segments are then exposed to enzymes such as collagenase to digest the matrix, thereby releasing the progenitor cells resident in the perivascular region. Following extraction, the isolated progenitor cells can be expanded and/or induced to differentiate into desired cell types using established cell culturing techniques. The progenitor cells derived from the perivascular region of blood vessels and particularly from umbilical cord vasculature including human umbilical cord vasculature show great promise as agents useful for the therapeutic regeneration and engineering of damaged or diseased tissue. It would be useful to improve upon the methods by which such cells are obtained.

SUMMARY OF THE INVENTION

In one of its aspects, the present invention provides a method for processing an umbilical cord to facilitate extraction of progenitor cells from the perivascular region thereof, the method comprising the steps of:

a) anchoring an excised umbilical cord at a location along the length thereof; b) cutting the cord at a site proximal to the anchored location to penetrate the epithelium while leaving the internal vasculature intact; and

c) removing the epithelium from the length of the cord by peeling the epithelium from the incision site and away from the anchored location, thereby providing exposed intact cord vasculature for subsequent processing.

The simple expedient of forming a cut on an anchored cord in one embodiment facilitates removal of the intact epithelium from the entire length of excised cord in one pulling motion, and significantly reduces the time and effort required to process a single cord, relative to prior art procedures, (should we include some language around the epithelium not coming off all in one piece?)

This method can be performed using an apparatus that provides a surface or support means for supporting the excised cord, desirably along its entire length. Anchoring means, associated with the support means, is provided to releasably engage the cord at a location along the length thereof, preferably at one end of the cord. Desirably, tension adjustment means may be provided in association with the anchoring means so that the pressure with which the cord is held by the anchoring means can be adjusted so that the cord is retained without being torn away when the epithelium is pulled therefrom. After the cord is "skinned" in this manner, individual blood vessels within the cord can be readily separated from each other and processed individually to extract progenitor cells from their external surfaces. To facilitate such processing, excess connective tissue can be removed manually from the vessel surface if desired. Furthermore, to avoid contamination by cells residing within the vessel or its endothelium, the open ends of the vessel desirably may be closed by clamping or otherwise ligatured such as by suturing before the vessel is processed further to extract cells from the perivascular region.

These and other aspects of the present invention are described in greater detail with reference to the accompanying drawings, in which: Figure 1 is a plan view of an apparatus suitable for "skinning" umbilical cord, showing the cord in place and in the process of being skinned; and

Figure 2 is a sectional view of the apparatus taken along lines II-II of Figure 1.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods and apparatus useful to improve the efficiency with which progenitor cells are extracted from the perivascular region of blood vessels. In embodiments, the blood vessels are umbilical cord blood vessels. In a preferred embodiment, the umbilical cord blood vessels are human umbilical cord blood vessels. In the alternative, the blood vessels are umbilical cord blood vessels obtained from non- human mammalian subjects including pets or livestock, particularly including horses, cattle, swine, sheep, and the like.

The "perivascular region" from which the desired progenitor cells are extracted comprises that region that is associated intimately with the external surface of the blood vessel. As used herein, the term "progenitor cells" refers to cells that will differentiate under controlled and/or defined conditions into cells of a given phenotype. Thus, an osteoprogenitor cell is a progenitor cell that will commit to the osteoblast lineage, and ultimately form bone tissue when cultured under conditions established for such commitment and differentiation. Umbilical cords useful in the present method can be obtained using well established practices. In one embodiment, an intact and full length cord (about 40cm) is collected post partum, for instance from full-term infants (caesarian or natural births) immediately upon delivery. The cord is transferred into a sterile vessel containing preservation medium (e.g. α-MEM containing antibiotics), and processed promptly.

The method of the present invention can then be applied to expose the cord vasculature, and ultimately to permit extraction of progenitor cells from the perivascular region of isolated individual umbilical cord blood vessels.

More particularly, and while working on a suitable surface and within an aseptic environment, the cord is first anchored, desirably (but not necessarily) at one end thereof. The cord is anchored in a way to resist movement of the cord when a pulling motion is applied along the length of the cord to remove the epithelium, as described below. At a site proximal to the anchored end of the cord, and downstream of the anchoring means, a cut is introduced to a depth sufficient to penetrate the epithelium, while leaving the internal vasculature intact. The cut desirably is made transversely, and around the complete circumference of the cord. However, it will be appreciated "stripping" of the epithelium can be achieved using one or more cuts that are not entirely circumferential in their length or orientation. For example, two hemispherical cuts could be made, one to the front of the cord and one to the back of the cord, in two cutting steps. Moreover, the single cut could be made substantially circumferentially, leaving a small segment intact that can be torn away during the stripping process. In the alternative, but less conveniently, the cord could be cut transversely and then longitudinally, to provide "corners" of epithelium that could be grasped during the stripping motion. Furthermore, while the incision should preferably engage the entire circumference of the cord, it is of course not essential that the incision is precisely transverse to the cord, i.e., precisely perpendicular to the cord length - it may for instance be elliptical in its orientation. The cut itself can be introduced using a knife and preferably a scalpel, either alone or in combination with scissors of any other cutting instrument. Once the anchored cord has been cut, the entire epithelium can then be stripped therefrom by engaging the epithelium at a site that is near to and downstream of the incision, and then pulling the epithelium in the downstream direction with a force leading away from the anchored site and along the length of the cord. For the pulling motion, the epithelium can be engaged by hand, or more conveniently using forceps or hemostats, clamps, etc. Most desirably, the epithelium may be grasped at two points, opposite each other, to perform a two handed operation.

The parting of the epithelium from the endogenous vasculature and associated matrix can be facilitated by introducing a further incision, or a tear, running at least a short distance downstream from the circumferential incision, to initiate the peeling process.

An apparatus suitable for performing this "skinning" process is produced using materials amenable to sterilization, and generally comprises components adapted to accommodate and retain the cord during the process.

According to embodiments of the present invention, such an apparatus comprises support means to provide a work surface that will accommodate the length of the umbilical cord; generally up to about 50cm or more in length and, for convenience, from about 10cm up to about 25cm or more in width. In combination with the support means, there is provided anchoring means adapted to releasably engage the cord. In addition, the apparatus desirably also provides tension adjustment means, cooperating with the anchoring means, so that the tension with which the cord is retained by the anchoring means can be adjusted at least from (i) a cord engaging position that retains the cord during the pulling motion but does not crush the cord to cause tearing of the cord away from the anchoring means during the pulling motion, to (ii) a cord releasing position permitting the cord, and particularly the waste end of the cord, to be released and discarded as desired. It will be appreciated that such an apparatus can take many different but equally useful forms. One embodiment of such an apparatus is shown in Figures 1-2, to which reference is now made.

As shown in Figure 1 , an apparatus comprises support means which may be in the form of a table 10 formed of a metal such as steel or any other material that can be sterilized, such as other metals and certain plastic materials. The table 10 provides a surface 12 that in the preferred embodiment accommodates the entire length of the supported umbilical cord or such length thereof as is being processed 14.

Anchoring means and associated tension adjustment means are mounted at one end of the table 10. The anchoring means may comprise pegs 16 spaced to accommodate the width of the supported cord, which cooperate with retainer member 18 for example via mounting holes 20. Anchoring is achieved by applying pressure to retainer member 18 so that the supported umbilical cord 14 is firmly engaged between the table surface 12 and the retainer member 18 (Fig. 2). The table may further comprise friction means, such as engraved stippling, on its surface between the pegs and beneath the retainer member to enhance cord retention.

Tension adjustment means in the exemplary embodiment shown is provided by threads formed on pegs 16 in combination with nuts 22 that are threadably engagable by the pegs. Threading of the nuts 22 permits the user to apply pressure or tension at the desired level onto the retainer member, thereby engaging the supported cord with the desired force.

In use, one end of the fresh, excised length of umbilical cord is received between the pegs 16, and the retainer member 18 is moved into a cord engaging position by winding nuts 22 onto and down the pegs 16, until the cord is firmly engaged. With the cord so anchored, one or more incisions 24 are made circumferentially around and through the cord epithelium, to a depth that leaves the internal vasculature intact. With the incision made, the lip of the cord formed by the incision 24 is grasped, using forceps or clamps positioned at opposite points of the lip, and a pulling motion is applied in the direction indicated by the arrows in Figure 1, i.e., along the length of the cord and away from the retainer member 18, thereby stripping the epithelium from the cord. In the alternative, and as shown in Figure 1 , the procedure can be facilitated by introducing a further longitudinal cut or tear 26 running downstream down from the lip formed by incision 24, to initiate and guide peeling of the epithelium.

In practice, the apparatus can be placed during use on any frictional surface, such as a sterile non-skid silicone pad, to resist movement of the apparatus during use. Of course, the apparatus can also be mounted firmly onto, or incorporated into, any surface.

Should the peeling procedure allow small patches or sections of epithelium to remain attached to the cord, these can be removed manually.

Once the cord is peeled, it can be removed from the apparatus either simply by cutting the cord at the anchor or by disengaging the anchor simply by unwinding the nuts 22 to release the retainer member 18 so that the peeled cord can be disengaged. The apparatus is then available for re-use, following sterilization.

It will be appreciated that numerous other designs can be implemented to provide a device having the functional attributes of the apparatus embodiment depicted herein. For instance, it will be appreciated that an apparatus that is similar in design can be produced by altering the retainer member so that one end thereof is engaged on the table such as by hinge means, and the other end of the retainer member is adapted to be moveable from a cord-engaging position to a cord-releasing position. In principle, this design is reflected in the present figures, wherein one end of the retainer member remains fixed in position by engagement of a nut on the peg associated with one end of the retainer member, and the other end of the retainer member is manipulated by unwinding the nut associated with the peg at that end of the retainer member, to raise that end of the retainer member to receive the cord. The cord is thus received, retained and released in a one-handed operation by manipulating only one of the two nuts shown in Figure 1. Similar one- handed designs are also within the scope of the present invention. The cord peeling process yields the cord vasculature with associated matrix, and is processed further to prepare the vessels for perivascular progenitor cell extraction. This is achieved by isolating individual vessels. Vessels can be isolated by first visualizing their location within the matrix at the sectioned end of the peeled cord, and then introducing incisions that run parallel to them so that they are then splayed individually from within the cord. Thereafter, each vessel is removed by teasing it away from the other vessels, along the length of the cord. Sections of excised vessel are also suitable. Excess matrix coating each vessel can also be removed manually, if desired.

The excised, individual blood vessels are then ready as substrates for progenitor cell extraction. Suitably, and to ensure that progenitor cells are extracted only from the perivascular region that lies on their external surfaces, the open ends of the vessels can be closed using sutures or clamps to avoid contamination from cells resident within their internal surface or space. The vessels also can be flushed to remove residual blood and blood components.

In this state, the vessels can either be processed promptly, or prepared cryogenically and stored frozen until needed. A particularly useful method for preserving the blood vessels in cryogenic storage, to ensure the subsequent recovery of viable cells therefrom, is described in co-pending WO007/071048 published June 27, 2007, incorporated herein by reference.

Briefly, cryogenic storage and subsequent recovery of thawed blood vessels proceeds as follows: The vessel or a vessel segment (1 vessel per tube, to avoid crowding) is placed in 10% DMSO and 90% FBS (cooled @ 4⁰C) in a 5OmL centrifuge tube, 5mL per 20cm vessel. In place of the 90% FBS, different serum-containing mixtures or blends can be used, such as 10% serum (FBS or other) and 80% DMEM or other media suitable for cell culturing. The tubes containing the vessel segment in the DMSO/serum solution are then cooled transitionally by first incubating for 30 minutes in a fridge at 4⁰C. The tubes are then removed, and the vessels are transferred to cryotubes/freezing bags. One vessel segment is added to each tube, or all vessels are transferred into a cryobag. To the cryotubes containing the vessels is added enough cryoprotectant solution to fully submerge the tissue (90% Serum, 10% DMSO as above). The contained vessel segments are then placed in a controlled rate freezer overnight (about 6-12 hours), reducing and holding the temperature to about -7O⁰C. Thereafter, the containers are transferred to liquid nitrogen storage (vapour phase), labeled for identification, and maintained at - 196⁰C until required.

To thaw the vessels, the containers are removed from the liquid nitrogen storage. The vessels are removed from the containers, and allowed to thaw for 10 minutes in a 37⁰C waterbath. The vessels are then transferred to 5OmL tubes, and washed in cooled (4C) PBS, using enough to double the cryoprotectant volume as determined earlier The tubes with vessel segments in the PBS are then allowed to incubate in the fridge at 4⁰C, to remove DMSO. The amount of cool PBS in each tube is then doubled, and the tubes are incubated for an additional 5 minutes in the fridge.

Following this procedure, the vessel segments are ready to serve as a source of viable progenitor cells.

To extract progenitor cells from prepared vessels that are either fresh or previously frozen as just described, published techniques can be applied. Briefly, and as described in co- pending WO2004/072273 published August 26, 2004 and US2005/0148074 published July 7, 2005 and by Sarugaser et al in Stem Cells, 23, 220-229 (2005), the vessel or vessel segment is subjected to enzymatic digestion to dissolve matrix and liberate the progenitor cells from the perivascular region. The resulting extract is then refined using various techniques including centrifugation to remove contaminants, and the isolated cells are cultured to expand their population.

More particularly, enzymatic digestion to release the progenitor cells from the surface of the blood vessels, or segments thereof, can proceed overnight in 5OmL lots with gentle mixing e.g., on a rotisserie apparatus, and in a collagenase solution (e.g., 0.1 to lOmg/mL collagenase). Upon removal from the digest, the cells are rinsed in ammonium chloride to lyse any red blood cells from the cord blood. Following this, the cells are rinsed and plated out in 85% α-MEM containing 5% fetal bovine serum and 10% antibiotics (penicillin G at 167 units/ml; Sigma, gentamicin 50 μg/ml; Sigma, and amphotericin B 0.3 μg/ml) at a density of 4,000 cells/cm². The cells are passaged when they reach 75- 80% confluence, which is approximately every 6-7 days.

Improvements in this process have now also been identified. According to embodiments of the invention, the vessels are more aggressively physically dissociated in the enzymatic solution, thereby reducing the necessary incubation time and increasing cell yield by decreasing cell death. This physical "tumbling" movement is superior to mixing in a rotisserie, as the added movement results in digested tissue ~ 5 times faster. Such physical movement can be achieved using a tumbling rotator device having sufficient motion to disturb the vessels aggressively. The digestion takes place at a temperature that is optimal for the digesting enzymes, such as 37C. In another preferred method, more than one enzyme can be used to not only release the desired cells from the collagen matrix around the vessels but also reduce the viscosity of the solution to make the processing time of the cells an order of magnitude faster (from 3 hours to 20 minutes). Thus, in embodiments of the invention, digestion proceeds in the presence of an enzyme that digests hyaluronic acid, thereby to reduce the viscosity of the suspension that results from digestion with collagenase. Accordingly, the enzymatic digestion step desirably proceeds in the presence of both a collagenase and hyaluronidase (HAse). The HAse can be present in a viscosity-reducing concentration, such as 0.1-250U/ml.

Thus, in specific embodiments, the liberation of perivascular cells from the blood vessels is achieved by introducing the blood vessel or a segment thereof into a solution comprising 20-40 mL saline or equivalent, and then adding collagenase and hyaluronidase to digest matrix and reduce viscosity of the digest suspension. After 2-4 hours of aggressive tumbling at 37C on a rotator device, the supernatant containing the cells is transferred and centrifuged, and the cells contained in the pellet are cultured either for expansion or under conditions suitable for inducing their differentiation. The perivascular progenitor cells isolated from human umbilical cord, now known by the acronym HUCPVCs, show great promise as agents useful in tissue regeneration and will be useful therapeutically to treat or replace damaged, diseased or missing tissue, as well as in immunological indications such as autoimmune diseases and Graft vs. Host Disease. By simplifying the procedure used to obtain the source of such cells, i.e., the umbilical cord blood vessels, the present invention facilitates the commercialization of such umbilical cord progenitor cells and indeed, any other useful cells or tissues that can be extracted from blood vessels including umbilical cord blood vessels.

Claims

WE CLAIM:

1. A method for processing umbilical cord, comprising the steps of: a) anchoring an excised umbilical cord at a location along a length thereof;

b) cutting the cord at a site proximal to the anchored location to penetrate an epithelium while leaving internal vasculature intact; and

c) removing the epithelium by peeling the epithelium from the cut, along the length of the cord, and away from the anchored location, thereby providing exposed intact cord vasculature for subsequent processing.

2. The method according to claim 1, wherein the cutting step introduces at least a transverse cut around the circumference of the cord.

3. The method according to claim 1, comprising the further step of introducing at least a further cut that runs longitudinally from the circumferential cut thereby to initiate and guide the peeling of the epithelium.

4. The method according to claim 1 , wherein the umbilical cord is human umbilical cord.

5. The method according to claim 1, wherein the umbilical cord is a non-human umbilical cord.

6. The method according to claim 1, wherein individual blood vessels are isolated from the umbilical cord so processed.

7. The method according to claim 1 , wherein said method is performed using an apparatus comprising:

a) support means for supporting the excised cord;

b) anchoring means, associated with the support means, adapted to releasably engage the cord at a location along the length thereof, and c) tension adjustment means, association with the anchoring means, adapted to adjust the tension with which the cord is engaged by the anchoring means.

8. An apparatus adapted to receive and process umbilical cord to facilitate removal of an epithelium therefrom, the apparatus comprising a) support means for supporting the excised umbilical cord; b) anchoring means, associated with the support means, adapted to releasably engage the cord at a location along a length thereof, and c) tension adjustment means, association with the anchoring means, adapted to adjust a tension with which the cord is engaged by the anchoring means.

9. A process for obtaining blood vessels useful as a source of perivascular progenitor cells, the process comprising the steps of: a) obtaining umbilical cord vasculature by the method according to any one of claims 1-6; and b) isolating individual blood vessels therefrom.

10. The process according to claim 9 wherein the isolated vessels are stored cryogenically before being treated to extract said progenitor cells.

11. The process according to claim 9, wherein said umbilical cord vasculature is human umbilical cord vasculature.

12. A process for obtaining progenitor cells, comprising the steps of: a) obtaining a blood vessel by the process according to any one of claims 9-11; and b) treating the blood vessel to extract progenitor cells from the perivascular region of said vessel.

13. A process for obtaining progenitor cells from a perivascular region of an umbilical cord blood vessel, comprising the steps of:

1) obtaining an isolated umbilical cord blood vessel; and

2) treating said blood vessel to release progenitor cells from the perivascular region thereof, wherein said blood vessel is treated using: a) A combination of collagenase and hyaluronidase; and/or b) Aggressive physical agitation of the blood vessel.

14. A process according to claim 14, wherein the blood vessel is obtained by the process according to any one of claims 8-10.