TWI586804B - Cryopreservation of umbilical cord tissue strips for cord tissue-derived stem cells - Google Patents

Cryopreservation of umbilical cord tissue strips for cord tissue-derived stem cells Download PDF

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Publication number
TWI586804B
TWI586804B TW105104858A TW105104858A TWI586804B TW I586804 B TWI586804 B TW I586804B TW 105104858 A TW105104858 A TW 105104858A TW 105104858 A TW105104858 A TW 105104858A TW I586804 B TWI586804 B TW I586804B
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Taiwan
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umbilical cord
cord tissue
tissue
method
umbilical
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TW105104858A
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Chinese (zh)
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TW201730333A (en
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曾珮娸
許祥瑞
黃啟軒
羅瑋瑜
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生寶生物科技股份有限公司
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Description

Cryopreservation of umbilical cord tissue and method for obtaining derived stem cells thereof

The present invention is broadly related to stem cells, and more particularly to a method of preserving umbilical cord tissue-derived stem cells.

There have been many documents pointing to the potential benefits of preserving a baby's cord blood. There is a similar concept for preserving umbilical cord tissue in a baby's umbilical cord segment at birth, where the cord tissue contains cells that may be available for subsequent use. The umbilical tissue can be frozen in a low temperature storage cabinet for long-term preservation. When the infant cells are needed for treatment in the future, the umbilical cord tissue can be treated to extract the cells therein using the best techniques available at the time.

For example, U.S. Patent Publication No. 2009/0275127 A1, the entire disclosure of which is incorporated herein by reference in its entirety, is incorporated herein by reference in its entirety in its entirety in its entirety in the the the the the the In the disclosed technique, an umbilical cord tissue is a blood vessel containing Wharton's jelly around the blood vessel, and the extracted precursor cells are human umbilical cord perivascular cells (HUCPVCs). In the disclosed method, a complete umbilical cord blood vessel with a Watson's gel associated therewith is obtained by slowly pulling the blood vessels away from the umbilical cord that has been longitudinally cut, and is laterally cut to produce Blood vessel fragment. Most of the Watton's gel in the umbilical cord will be removed during this process, but the Wyatt's gel that is associated with the extracted blood vessel in the peripheral region of the blood vessel is left behind. The end of the umbilical blood vessel is tied off to avoid any residual blood leakage from the blood vessel. On the other hand, blood in the blood vessels can be removed by repeated rinsing.

A method of preserving an umbilical cord is disclosed in U.S. Patent No. 8,703,411 B2, which is incorporated herein by reference. The disclosure of this patent is incorporated herein by reference in its entirety. The method disclosed in the patent comprises obtaining an umbilical cord segment; the umbilical cord is chopped into a plurality of umbilical tissue fragments; and the umbilical tissue fragments are mixed with an antifreeze solution containing one antifreeze and one protein to form a mixture. Shaking the mixture for a period of not less than 20 minutes and not more than 40 minutes; and cryopreserving the mixture.

The preservation of umbilical cord tissue and subsequent cell culture are relatively novel techniques. While the prior art provides a viable method of preserving umbilical cord tissue, there is still a need for an efficient method of preserving umbilical cord tissue for subsequent use.

The invention relates to a method for cryopreserving umbilical cord tissue. According to an embodiment of the invention, the method comprises the steps of: (a) applying tension to an umbilical cord segment to expand a gap between three umbilical cord blood vessels in the umbilical cord segment; b) cutting the umbilical cord segment in a longitudinal direction parallel to the length of the umbilical cord blood vessel to obtain a umbilical cord tissue strip, wherein the strip of umbilical cord tissue comprises the umbilical cord blood vessel Wharton's jelly, which is derived from a perivascular region, an intervascular region, and a subamnion region; (c) The strip-shaped umbilical cord tissue is inocubating with a cryogenic composition; and (d) cryopreserving the strip-shaped umbilical cord tissue containing the Watton's gel and the low temperature composition.

According to an embodiment of the invention, the method further comprises longitudinally cutting the remainder of the umbilical cord segment to obtain two additional strips of umbilical cord tissue.

According to an embodiment of the invention, the culturing step is performed as soon as possible after shearing, for example between 5 and 10 minutes after the first dicing, and preferably at the 6th minute after the first dicing. .

According to an embodiment of the invention, the method further comprises applying tension to the umbilical cord segment using a tool or a probe. The tool or one end of the probe can be inserted into an umbilical blood vessel while the other end is fixed or pivotally connected to a stationary device.

According to an embodiment of the invention, the method does not involve digesting the strip of umbilical cord tissue with any enzyme.

According to an embodiment of the invention, step (c) further comprises shaking the strip of umbilical cord tissue in the low temperature composition for 20 to 40 minutes.

Another aspect of the invention relates to a method of obtaining umbilical cord tissue-derived stem cells. According to an embodiment of the invention, the method comprises the steps of: (a) thawing a cryopreserved strip of umbilical cord tissue, wherein the cryopreserved strip The umbilical cord tissue comprises a umbilical cord tissue and a low temperature composition, the strip umbilical cord tissue comprising an umbilical cord blood vessel and a Watson's gel, the Watson's gel comprising a peripheral region of a blood vessel, an intervascular region, and a gelatinous tissue in the submucosal region; (b) removing the cryogenic composition; (c) cutting the strip of umbilical cord tissue to form a plurality of umbilical tissue fragments, wherein the umbilical tissue fragments are larger than 2 And (d) cultivating the umbilical tissue fragments in a medium to obtain the umbilical cord tissue-derived stem cells. The umbilical tissue fragment size is greater than 2 mm and cannot pass through a screen having a 2 mm opening. According to an embodiment of the present invention, the umbilical tissue fragment has a size of not less than 0.5 cm (i.e., 0.5 cm or more).

According to an embodiment of the invention, the method for obtaining umbilical cord tissue-derived stem cells comprises not treating the strip of umbilical cord tissue with an enzyme.

The embodiments of the present invention are described by way of example only. Other equivalent modifications or variations may be made by those of ordinary skill in the art without departing from the scope of the invention. Various embodiments of the present invention are described in detail in the following paragraphs, and elements in the reference drawings are denoted by the numerical symbols.

definition

The terms used in this specification have their ordinary meaning in the art, the context of the invention, and the specific context. The specific terms used to describe the invention are discussed below, or in other portions of the specification, to provide additional guidance in the practice of the invention. For convenience, specific terms will be marked in bold, italic, and/or quotation marks. Bold marks have no effect on the scope of the invention and the meaning of the terms; the definition of terms is the same in the context of whether or not there is a bold mark.

Unless otherwise defined, those of ordinary skill in the art will understand that the technical and scientific terms used herein have the same meaning as the ordinary terms. In case of conflict, this document and the definitions shall prevail. It is understandable that the same thing can be recited in more than one way, so one or more terms may be represented by alternative words or synonyms when discussing this document; it is not of special significance whether a term is to be elaborated or discussed in this article. Synonyms may be used in a particular term and are not excluded from the use of other synonyms when describing one or more synonyms. The use of any examples in this specification, including any terms discussed herein, is for illustrative purposes only and does not limit the scope or meaning of the invention, or any interpretative terms. Also, the present invention is not limited to the following embodiments.

The terms "about", "about", or "probably" mean within 20% of a value or range, more preferably within 10%, and most preferably within 5%. The numbers described herein are approximate, that is, if not explicitly stated, the terms "about", "about", or "probably" can be used for inference. In addition, any numerical range disclosed herein includes all values within the range, and each value in the equivalent range is separately disclosed.

The terms "an umbilical cord tissue piece", "cord tissue pieces", and "coarse cord tissue pieces" are interchangeable. A rough umbilical tissue fragment means a piece of umbilical cord tissue having a size greater than 2 mm, preferably greater than 3 mm in size, more preferably greater than 4 mm in size, and more preferably greater than 5 mm in size. An umbilical tissue fragment having a size greater than 2 mm means that the umbilical tissue fragment cannot pass through a cell filter having a mesh size of 2 mm.

The term "a segment of an umbilical cord" means a part of an umbilical cord. The length of an umbilical cord segment may be any length between 0.5, 1, 2, 3, 5, 10 centimeters, longer, or longer.

The phrase "cryopreservation or cryopreserving" means a process in which cells or whole tissues are cooled to a temperature below freezing, such as 77 K or -196 ° C (boiling point of liquid nitrogen). At such low temperatures, it will effectively arrest any biological activity, including biochemical reactions leading to cell death. However, when the antifreeze solution is not used, the cell preservation often causes damage when it is cooled down to freezing or thawing to room temperature.

The terms "freezing" and "cryopreserving" are used interchangeably. The terms "cryogenic composition", "cryogenic solution", "cryopreservation composition", and "anti-freezing solution" are used interchangeably. . Any low temperature composition or cryopreservation composition is commonly used in the art. For example, a low temperature composition or a cryopreservation composition may contain dimethyl sulfoxide (DMSO) at a concentration of about 1% to 70%, preferably at a concentration of about 5% to 55%. Other examples of low temperature compositions or cryopreservation compositions, such as the method of U.S. Patent No. 8,703,411 B2, may comprise an antifreeze and a protein to form a mixture.

The terms "cord tissue-derived cells", "cord tissue pieces-derived cells", "cord tissue-derived stem cells", and "umbilical cords" Tissue-derived MSCs are interchangeable unless otherwise specified.

The term "doubling time" means the time required to double the volume or value.

Example

Exemplary instruments, devices, methods, and related experimental results are described below in accordance with embodiments of the present invention without limiting the scope of the invention. The headings and subtitles appearing in the examples are for ease of reading and do not limit the scope of the invention. In addition, the specific theory presented and disclosed herein is not to be construed as limiting the scope of the invention.

Materials and Methods

A method of preparing a control sample is disclosed in U.S. Patent No. 8,703,411 B2. Briefly, an appropriate length of umbilical cord is cleaned and sterilized. After removing the two umbilical cord arteries, the non-directional shearing umbilical cord tissue becomes a piece of about 2 mm each. It is the most ideal time to apply the enzyme fragments to the enzyme treatment. When the enzyme action is terminated, the tissue fragments are washed to remove residual enzymes, then the antifreeze solution containing DMSO is added, and the tissue fragments are placed in liquid nitrogen for storage. After cryopreservation for more than one week, the tissue fragments are thawed and washed to remove the antifreeze solution for primary cell culture. Cell collection, cell number calculation, and cell viability and characterization assays were performed according to cell growth conditions.

The method disclosed in U.S. Patent No. 8,703,411 B2 is time consuming because the umbilical cord is smooth and difficult to cut. The method of the present invention is designed to overcome the slip and toughness of the umbilical cord and is conveniently handled by only one person.

In accordance with an embodiment of the present invention, a tool is provided that provides support and retention, such as pliers, that can be inserted into the umbilical cord without the need to remove blood vessels within the umbilical cord. In addition, a fixture (or fixture) can secure the umbilical cord in place and stretch open the umbilical cord so that the umbilical cord tissue can be conveniently cut longitudinally by a single person.

An umbilical cord consists of three blood vessels, one vein and two arteries. According to the method of the present invention, an umbilical cord can be longitudinally cut into two or three strip-shaped umbilical cord segments, each segment comprising at least one blood vessel and a Watson's gel around the blood vessel to which it is associated, wherein the Watton's gel is from In a peripheral region of a blood vessel, an intervascular region, and an amnion region. Therefore, according to an embodiment of the present invention, there is a method of cutting two to three knives to obtain a strip of umbilical cord tissue which is completely exposed by the Wharton gel layer, so that the Wharton gel layer can be directly stored with the cryopreservation solution. Contact, thereby increasing the penetration efficiency of the cryopreservation solution into the Watson's gel layer. The cryopreservation solution can be directly added to the strip umbilical cord tissue. After mixing with the cryopreservation solution, the umbilical strip tissue can be stored in liquid nitrogen.

To demonstrate the cellular activity preserved by the method of the invention, after cryopreservation for more than one week, the strip tissue is thawed, washed to remove the antifreeze solution, and sheared to pieces each larger than 2 mm in size (eg: 2~10 mm or 2~5 mm is preferred), and then the facet of the Wharton gel layer is placed face down for primary culture (ie towards the bottom of the dish). Cell collection, cell number calculation, and cell viability and characterization assays (surface marker analysis and cell viability assay) were performed according to cell growth conditions.

According to the cell dynamics colony forming unit (CFU), a control group (method according to U.S. Patent No. 8,703,411 B2) and an experimental group (method of the present invention) were collected after 10 to 14 days. Primary umbilical cord mesenchymal stem cells. Staining with 7-aminoactinomycin-D (7-AAD), and analyzing the positive and negative surface markers of mesenchymal stem cells, stage-specific embryonic antigen 4 by flow cytometry (stage-specific Embryonic antigen 4, SSEA-4) [a cell surface glycosphingolipid gene], and cell viability.

Embodiment 1 Preparation of strip umbilical cord tissue:

Figure 1 is a schematic illustration of a fixation device 120 that can be used to shear an umbilical cord into a strip of umbilical cord tissue prior to fixation. This is an embodiment of the device that makes the method of the invention easier to accomplish. Those of ordinary skill in the art understand that other devices can be used as long as the method of the present invention can be made easier to operate. As shown in Fig. 1, in the embodiment, the fixture 120 is provided with two slots (or holes) 1 and 2 for securing/accommodating tools (e.g., pliers) for umbilical cord shearing by the method of the present invention.

Fig. 2A is a schematic view showing a cross section of an umbilical cord having three blood vessels. An umbilical cord consists of three layers: the amnion, the umbilical cord blood vessels (ie, the two arteries and one vein), and the matrix (a mucous connective tissue called the Wharton gel, which is located between the amniotic epithelial cell lining and the umbilical cord blood vessels). Watson's gel can be subdivided into three distinct regions: the subarachnoid region (about the area lining the amniotic epithelial cells), the intervascular region (about the region between the umbilical vessels), and the perivascular region ( It is roughly around the blood vessels of each umbilical cord).

Figure 2B illustrates the concept of one embodiment of the present invention. As shown in FIG. 2B, an umbilical cord 100 contains a vein 102 and two arteries 104 and 106. The three cutting lines shown are located between the umbilical cord blood vessels, which can separate three blood vessels into three strips of umbilical cord tissue, and are surrounded by Watson's gel. Each strip of umbilical cord tissue contains an umbilical cord blood vessel and a Wyton's gel, and the Wharton gel obtained therefrom is derived from three regions: the submucosal region, the intervascular region, and the peripheral region of the blood vessel. In this embodiment, the vein 102 forms a strip of umbilical cord tissue 110 with the surrounding Watton gel layer. The artery 104 forms another strip of umbilical cord tissue 112 with the surrounding Watton gel layer. The artery 106 forms another strip of umbilical cord tissue 108 with the surrounding Watton gel layer.

Once the strips of umbilical cord tissue are separated, more of the surface of the Watson's gel will be exposed, as shown in Figure 2C. The exposed surface area of the larger Wharton gel promotes the penetration of the cryopreservation solution into the strip of umbilical cord tissue. As a result, cells within the strip umbilical cord tissue are less likely to be damaged during freezing and thawing, resulting in more available cells being recovered from the cryopreserved strip of umbilical cord tissue.

The method of the present invention is designed to rapidly separate the umbilical cord while increasing the contact area of the Watson's gel layer with the low temperature solution. It can be used with a supporting and fixing tool (such as a pair of pliers) to fix and maintain the umbilical cord.

For example, in accordance with Figures 2A through 2C, one end of a tool, such as a pair of forceps, is inserted into a lumen of a blood vessel (e.g., umbilical vein 102) within an umbilical cord segment 100. Next, the other end of the same tool can be secured to the cassette device, such as into a slot (or hole) 1 in the umbilical cord fixture 120 (Fig. 1). In a similar procedure, the posterior end of the second instrument is inserted into the lumen of the second vessel (eg, umbilical artery 104). The other end of the second tool is secured in slot 2 on the umbilical fixture 120 (Fig. 1).

Similarly, one end of the third tool is inserted into the lumen of the third blood vessel (eg, umbilical artery 106). Next, a slight tension is applied to the umbilical cord (through the insertion tool) to dilate the voids with other blood vessels, such as the umbilical artery 106, the umbilical vein 102, and the umbilical artery 104.

When the umbilical cord is under the tension, a shearing tool (such as a scalpel) can be used to cut the area of the Watson's gel, and longitudinally cut along the gap between the umbilical vessels, such as the umbilical artery 106 and the umbilical vein 102. The cutting direction is parallel to the length of the umbilical blood vessels. Next, the second knife is longitudinally cut along the gap between the umbilical artery 106 and the umbilical artery 104 to obtain a strip of umbilical cord tissue 108 comprising the umbilical artery 106.

Likewise, strip umbilical tissue 110 and 112 can be obtained by subsequent shearing. For example, when tension is applied to the remaining umbilical cord to open the Watson's gel between the umbilical vein 102 and the umbilical artery 104, further cutting the Watson's gel separates the remaining umbilical cord into two strips of umbilical cord Organizations 110 and 112.

Of particular note that the separation of the insertion tool and the umbilical cord described in the above examples requires a specific sequence. Without departing from the scope of the invention, those of ordinary skill in the art will understand the order of the insertion tool and the separation of the umbilical cord. In addition, when the tool is inserted into the lumen of the blood vessel as described above, it is still possible to insert a Watson's gel layer close to the blood vessel. It will be appreciated by those of ordinary skill in the art that the inserted tool provides a slight tension to open the space between the blood vessels and/or straighten the umbilical segment to facilitate the cutting operation. Thus, the tool can achieve the same operational purpose by insertion into the lumen of a blood vessel or insertion into a Watson's gel layer close to the blood vessel.

After obtaining the strips of umbilical cord tissue, the strips of umbilical cord tissue will be washed, disinfected, and immersed in a cryopreservation solution to prevent damage to the cells during freezing or thawing. For example, the strips of umbilical cord tissue 108, 110, and 112 are placed in a test tube containing an antifreeze solution for a suitable period of time (eg, 30 seconds to several minutes) to clean the strip of umbilical cord tissue. The strips of umbilical cord tissue are then washed with a buffer solution to remove impurities.

The strips of umbilical cord tissue 108, 110, and 112 are then mixed and cultured with a cryopreservation solution at an appropriate time (e.g., 20-60 minutes) at a low temperature (e.g., below 10 ° C) and shaken. The umbilical cord tissue and the cryopreservation solution should be rapidly mixed and cultured after shearing, for example, mixed culture at 5, 6, 7, 8, 9, or 10 minutes after the first cut; It is better within 6 minutes after cutting. Finally, the strips of umbilical cord tissue 108, 110, and 112 can be slowly cooled to a suitable temperature (e.g., -70 °C) and then stored at a cryogenic storage temperature (e.g., below -120 °C) in a liquid nitrogen drum.

Figure 3 is a flow chart illustrating one of the embodiments of the present invention for preparing a strip of umbilical cord tissue for cryopreservation. One of the appropriate lengths of the umbilical cord segment (for example: about 3 cm, 5 cm, 7 cm, or 10 cm, preferably 3-7 cm) contains three umbilical cord blood vessels. As described above, one or more tools are inserted into the umbilical vascular lumen. Applying a mitigating force to stretch open the Watton's gel layer (first space) between the first and second and third blood vessels, and then longitudinally cutting the first space (ie, parallel) with a scalpel In the blood vessels, a first strip of umbilical cord tissue (the Wadden gel layer containing the first blood vessel and its associated knot) is obtained. Thereafter, the Watson's gel layer (second space) between the second and third blood vessels is opened, and the second space (parallel to the blood vessels) is longitudinally drawn to obtain a second strip of umbilical cord tissue (including There is a second blood vessel and its associated Watton's gel layer) and a third strip of umbilical cord tissue (including the third vessel and its associated Watton's gel layer).

4A and 4B are photographs showing the difference between the present invention (Fig. 4B: experimental group) and the prior art (Fig. 4A: control group, refer to U.S. Patent Publication No. 8,703,411 B2) for preparing umbilical cord tissue for cryopreservation.

Figure 4A (Prior Art) illustrates disinfecting, washing, removing two of the arterial vessels, and manually and non-directionally shearing the umbilical cord tissue to pieces each about 2 mm in size. Then, the pieces are subjected to an enzyme digestion treatment for about 20-40 minutes, after which the digestion solution is added to a serum-containing medium or an enzyme terminating reagent to terminate the digestion of the enzyme. After the pieces are washed in a buffer or cell culture medium (such as dulbecco's modified eagle medium, DMEM), the tissue fragments are precipitated in a low speed centrifuge and the residual enzyme is removed. The tissue pieces were then shaken and mixed with the cryopreservation solution at 10 ° C for 20-60 minutes, followed by storage at an ultra-low temperature in liquid nitrogen.

To test the activity of cryopreserved cells, after more than one week of cryopreservation, the same in the tissue fragments was quickly thawed at 37 °C, washed several times, and then the pellets were precipitated in a low-speed centrifuge to complete Remove the cryopreservation solution. Next, the tissue fragments were subjected to primary cell culture, and cells were collected after 10-14 days depending on the cell growth condition.

4B illustrates an embodiment of the present invention in which an umbilical cord segment is sterilized, cleaned, and cut into elongated strip-like umbilical cord tissue without first removing the umbilical cord blood vessel. Next, the strip of umbilical cord tissue is mixed (e.g., shaken) with the cryopreservation solution for a period of time (e.g., 20-60 minutes, preferably 30-50 minutes) at a suitable temperature (not greater than 10 °C, such as 0-10 °C). The strip of umbilical cord tissue is then stored in an ultra-low temperature (eg, in liquid nitrogen).

To test the cell viability of cryopreservation, after cryopreservation for more than one week, the same fraction of the tissue fragments was rapidly thawed at 37 °C and washed several times, and the fragmented tissue was precipitated in a low speed centrifuge. After the cryopreservation solution is completely removed, the strip of umbilical tissue will be cut into pieces (thick umbilical tissue fragments) each having a size greater than 2 mm (eg, 5 mm) with a sharp tool (eg, scissors or scalpel). The massive tissue was subjected to primary cell culture, and cells were collected after 10-14 days depending on the cell growth condition.

Figure 4C is a photograph illustrating the difference in size of umbilical tissue fragments 704 (obtained according to the method of Figure 4A) and rough umbilical tissue fragments 702 (obtained according to the method of Figure 4B).

Figures 5A through 5C illustrate various examples of securing devices for securing an umbilical cord to facilitate a shearing operation in accordance with an embodiment of the present invention. The fixture is designed for simplicity, convenience, single operation, and minimal contamination opportunities. In FIG. 5A, the fixture 5 includes the following components: a base 51, a bracket 52, a post 53 and a slot 54.

(i) The base 51 has a top surface 511, a bottom surface 512, a front end 513 and a rear end 514. The top surface 511 includes a plurality of ridges 515 adjacent the front end 513, and the ridges 515 are evenly spaced from each other. (being evenly spaced apart).

(ii) The bracket 52 has a top surface 521, a bottom surface 522, a front end 523 and a rear end 524. The top surface 521 includes one or more slots 525 near the front end 523, and each slot 525 can be One end of a tool or probe is suitably received and the rear end 524 can be coupled to the rear end 514 of the base 51.

(iii) The post 53 has a top end 531, a bottom end 532, a front surface 533 and a rear surface 534 that can be coupled to the bottom surface 522 of the bracket 52, and the bottom end 532 can be slidably engaged The top surface 511 of the base 51 is located between the ridges 515.

(iv) The housing 54 is movably coupled to the bottom surface 512 of the base 51.

The fixture 5 of Figure 5A is only one embodiment. Those skilled in the art will recognize that the fixture can be modified and varied in accordance with the methods of the present invention. for example. Figure 5B illustrates another example of a method of practicing the invention using the fixture. In FIG. 5B, the fixing device 6 comprises the following components: a base 61, a bracket 62, a connecting body 63, a hollow cap type rotating seat 64 and a nut 65.

(i) The base 61 has a top surface 611, a bottom surface 612, a front end 613 and a rear end 614, and is fixed to the top surface 611 of the base 61 by a screw (not shown), and a ball type A fixed base 615 is located above the screw.

(ii) The bracket 62 has a top surface 621, a bottom surface 622, a front end 623 and a rear end 624. The top surface 621 includes at least two slots 625 near the front end 623, and each slot 625 is suitable. One end of a tool (not shown) or a probe (not shown) is accommodated, and the bracket 62 has a screw hole 626.

(iii) the connecting body 63 has a spherical end 631 detachably coupled to the ball-shaped fixing base 615, and a threaded hole (not shown) opposite to the spherical end 631 The other end.

(iv) The hollow cap type rotary seat 64 has a top end 641 and a bottom end 642. The top end 641 and the bottom end 642 each have an opening. The top end 641 has a notch 643 connected to the top end 641. Opening, the hollow cap type rotary seat 64 is screwed and the spherical end 631 of the connecting body 63 is placed inside the hollow cap type rotating seat 64, and the top end 641 of the hollow cap type rotating seat 64 has an opening smaller than the spherical shape The end 631 is such that the hollow cap type rotating seat 64 can screw the spherical end 631, and the other end of the connecting body 63 protrudes from the top end 641 and the notch 643.

(v) The nut 65 has one end that passes through the screw hole 626 to screw the bracket 62.

Figure 5C illustrates another example of a method of practicing the invention using the fixture. In Figure 5C, the fixture 7 includes the following components: a base 71, a bracket 72, a transverse slot 73, and two vertical recesses 74.

(i) The base 71 has a hollow rectangular box structure without a bottom surface, and includes a top vertical wall 711 and two side walls 712 perpendicular to the top vertical wall 711. The two side walls 712 have a trapezoidal shape and Parallel to each other, the longer sides of the side wall 712 are combined with the top vertical wall 711 and the shorter side maintains the fixture perpendicular to the platform, and each side wall 712 has a beveled edge 713 to maintain the fixture 7 The slant stands on a platform 714.

(ii) The bracket 72 has a top surface 721, a bottom surface 722, a front end 723 and a rear end 724. The top surface 721 includes at least two slots 725 near the front end 723, and each slot 725 is suitable. One end of a tool (not shown) or a probe (not shown) is accommodated.

(iii) The lateral slot 73 is provided in the top standing wall 711 to fit the bracket 72.

(iv) Two vertical grooves 74 are provided inside the side wall 712 and extend downward from the lower side of the lateral slot 73 for fixing the bracket 72.

In order to understand the effect of "length of strip umbilical cord tissue" and "size of umbilical cord tissue obtained by thawing and shearing strip umbilical cord tissue" on cell culture, the applicant conducted different comparative experiments in which the strip umbilical cord The test length of the tissue ranges from 0.5 cm to 10 cm; and after thawing, the tissue fragments used for tissue culture range from 0.5 cm to 10 cm. Comparing the cell numbers and culture results of different groups: It was found that the stripe umbilical cord tissue with the length of 1-10 cm was the best. However, a length exceeding this range still yields good results, so the length of this range can also be used. In particular, the preferred strip umbilical cord tissue is 3-7 cm in length.

Embodiment 2 Comparison of the present invention with prior art methods:

Figure 6A is a result diagram comparing the operation time of the method of the present invention with the method of the prior art (i.e., U.S. Patent Publication No. 8,703,411 B2). This operating time was used to prepare cryopreserved umbilical cord tissue. The preparation method has been described in detail in the first embodiment. The present invention (experimental group) saves time, does not require removal of blood vessels (about 30 minutes), does not require enzyme treatment and terminates the enzyme reaction, and does not require washing to remove the enzyme (about 45 minutes). The new cutting method/method saves time in cutting the umbilical cord tissue (60 minutes for the control group and 10 minutes for the experimental group). Overall, the method of the present invention can save about 125 minutes over the prior art.

In contrast to the methods disclosed in the prior art, the present invention has one or more of the following advantages: simple, rapid, increased cell culture rate, increased primary cell culture yield, and reduced human factors.

Figure 6B is a graph showing the cell yield of the control and experimental groups. The umbilical cord tissue sources in both the control and experimental groups were from the same umbilical cord. Cell yield is calculated by dividing the total number of cells collected by the tissue weight of the primary cell culture (i.e., the number of cells per gram of tissue). The yield of the primary culture in the control group was 2.6 x 10 5 ± 2.5 x 10 5 cells (mean ± SD), while the experimental group was 1.3 x 10 6 ± 1.0 x 10 6 cells (mean ± SD). Using the two-sample t-test analysis results, the cell yield of the experimental group was 5 times that of the control group, and the p-value was less than 0.05 (the number of samples was 6) was statistically significant. In addition, the primary culture cell yield of the experimental group showed less variability between the samples compared with the control group (control group: CV% = 96.34%; experimental group: CV% = 77.93%)

Considering the time saved by the initial cell culture, the experimental group saved 1.8 days (Fig. 6C, control group: 12.83 ± 2.71 days; experimental group: 11 ± 0.89 days) (mean ± SD). The cell viability and purity of primary umbilical mesenchymal stem cells (UC-MSC) with good quality and no difference were obtained in both groups.

Figure 6D illustrates the results of analysis of cell surface antigen markers. The results are consistent with the International Society of Cellular Therapy (ISCT) guidelines for mesenchymal stem cells: positive markers (eg, CD13, CD29, CD44, CD73, CD90, CD105, and HLA-ABC) are greater than 95% And negative markers (such as CD31, CD34, CD45, and HLA-DR) are less than 2%. The early stem cell marker SSEA-4 was expressed in both groups and the expression was very similar. Primary umbilical cord mesenchymal stem cells were active in both groups greater than 95%.

Therefore, the experimental group is time-saving, simple, convenient, less dimensional difference caused by manual operation, avoiding or reducing mechanical damage caused by shearing tissue to fragments, and increasing the exposure of Watson's gel layer and The advantage of cryopreservation solution contact surface area. Frozen tissue viability is so high that subsequent processing can increase the yield of primary cultured cells to obtain umbilical cord tissue fragments.

However, the above-mentioned embodiments are merely exemplary embodiments of the present invention, and those skilled in the art can make the inventions of the present invention, and other equivalent modifications or changes can be made without departing from the scope of the invention. Accordingly, the scope of the invention is defined by the scope of the appended claims.

(120)‧‧‧Fixed devices

(1) ‧‧‧ slots (or holes)

(2) ‧‧‧ slots (or holes)

(100)‧‧‧Umbilical cord

(102)‧‧‧ vein

(104) ‧‧‧Arteries

(106) ‧‧‧Arteries

(108) ‧‧‧ strip umbilical cord tissue

(110) ‧‧‧ strip umbilical cord tissue

(112) ‧ ‧ strip umbilical cord tissue

S91-S98‧‧‧Steps

(702)‧‧‧Rough umbilical cord tissue fragments

(704) ‧ ‧ umbilical cord tissue fragments

(5) ‧‧‧Fixed devices

(51)‧‧‧Base

(511)‧‧‧ top surface

(512)‧‧‧ bottom surface

(513) ‧‧‧ front end

(514) ‧‧‧ Backend

(515) ‧ ‧ erection

(52) ‧‧‧ bracket

(521)‧‧‧ top surface

(522)‧‧‧ bottom surface

(523) ‧ ‧ front end

(524) ‧‧‧ Backend

(525) ‧‧‧ slots

(53) ‧‧‧ pillar

(531)‧‧‧ top end

(532)‧‧‧ bottom end

(533)‧‧‧ Front surface

(534) ‧‧‧Back surface

(54)‧‧‧Seat

(6) ‧‧‧Fixed devices

(61)‧‧‧Base

(611)‧‧‧ top surface

(612)‧‧‧Bottom surface

(613) ‧‧‧ front end

(614) ‧‧‧ Backend

(615)‧‧‧Spherical fixed base

(62) ‧‧‧ bracket

(621)‧‧‧ top surface

(622)‧‧‧ bottom surface

(623) ‧‧‧ front end

(624) ‧‧‧ Backend

(625) ‧‧‧ slots

(626)‧‧‧ Screw holes

(63)‧‧‧Connecting body

(631)‧‧‧Spherical end

(64)‧‧‧ hollow cap type rotary seat

(641)‧‧‧ top end

(642)‧‧‧ bottom end

(643) ‧ ‧ gap

(65)‧‧‧ Nuts

(7) ‧‧‧Fixed devices

(71)‧‧‧Base

(711)‧‧‧Top wall

(712)‧‧‧ Side wall

(713)‧‧‧ oblique sides

(714) ‧‧‧ platform

(72) ‧‧‧ bracket

(721)‧‧‧ top surface

(722)‧‧‧Bottom surface

(723) ‧‧‧ front end

(724) ‧‧‧ Backend

(725)‧‧‧ slots

(73)‧‧‧Horizontal slots

(74)‧‧‧Vertical slots

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic view showing an apparatus for fixing and cutting an umbilical cord in an embodiment of the present invention. Figure 2A is a schematic view showing a cross section of an umbilical cord. 2B is a schematic view showing the operation of preparing a plurality of strip-shaped umbilical cord tissues by an umbilical cord in the embodiment of the present invention. Figure 2C is a schematic diagram showing the contact of the cryopreservation solution with the strip of umbilical cord tissue. 3 is a flow chart showing the steps of processing an ultra-low temperature preservation of an umbilical cord in an embodiment of the present invention. Figure 4A is a photograph illustrating the treatment of an umbilical cord for cryopreservation, thawing, and culturing of the pre-stored umbilical cord to obtain primary umbilical cord mesenchymal stem cells (UC) in accordance with U.S. Patent No. 8,703,411 B2. -MSCs) method. 4B is a photograph illustrating a method of treating an umbilical cord for cryopreservation, thawing, shearing the umbilical cord to a coarse crumb, and culturing the fragment to obtain primary umbilical mesenchymal stem cells in accordance with an embodiment of the present invention. Figure 4C is a photograph comparing the size of the umbilical cord tissue as disclosed in U.S. Patent No. 8,703,411 B2 to the size of the umbilical cord tissue obtained by the method of the present invention. FIG. 5A is a schematic view showing a tool for fixing a position of an umbilical cord according to an embodiment of the present invention, which is processed in a convenient manner to form a strip-shaped umbilical cord tissue for cryopreservation. Fig. 5B is a schematic view showing a tool for fixing the position of the umbilical cord in another embodiment of the present invention, which is processed in a convenient manner to form a strip-shaped umbilical cord tissue for cryopreservation. Fig. 5C is a schematic view showing a tool for fixing the position of the umbilical cord in another embodiment of the present invention, which is processed in a convenient manner to form a strip-shaped umbilical cord tissue for cryopreservation. Fig. 6A is a graph showing the results of the operation of the experimental group (the method of the present invention) and the control group (method according to U.S. Patent No. 8,703,411 B2) before cryopreservation. Figure 6B is a graph showing the number of primary cultured UC-MSCs per gram of umbilical cord tissue in the experimental group (method of the present invention) and the control group (method according to U.S. Patent No. 8,703,411 B2). Figure 6C is a graph showing the number of days of primary culture in the experimental group (method of the present invention) and the control group (method according to U.S. Patent Publication No. 8,703,411 B2). Figure 6D is a table comparing the experimental group (the method of the present invention) with the control group (U.S. Patent Publication No. 8,703,411 B2), which is positive (negative) and mesenchymal stem cells (MSCs). The amount of surface factor expressed and cell viability.

S91-S98‧‧‧Steps

Claims (14)

  1. A method for cryopreserving umbilical cord tissue comprising the steps of: (a) applying tension to an umbilical cord segment to expand a space between three umbilical cord blood vessels in the umbilical cord segment; (b) a gap between the umbilical cord blood vessels along The umbilical cord segment is longitudinally cut parallel to the length of the umbilical cord blood vessel to obtain a umbilical cord tissue, wherein the strip umbilical cord tissue comprises the umbilical cord blood vessel and Wharton's jelly, the Watton's gel is derived from a blood vessel a peripheral region, an intervascular region, and an submucosal region; (c) mixing and culturing the strip of umbilical cord tissue with a low temperature composition; and (d) cryopreserving the strip of umbilical cord tissue containing the Watton's gel And the low temperature composition; wherein the method comprises not peeling, pulling away, or removing the umbilical blood vessels from the umbilical cord.
  2. The method of cryopreserving umbilical cord tissue according to claim 1, wherein before step (c), further comprising: (b') longitudinally cutting the remaining portion of the umbilical cord segment to obtain two additional strips of umbilical cord tissue.
  3. A method of cryopreserving umbilical cord tissue as described in claim 1 wherein the culturing step begins between 5 and 10 minutes after the first dicing.
  4. A method of cryopreserving umbilical cord tissue as described in claim 1, wherein the culturing step begins at the 6th minute after the first dicing.
  5. The method of cryopreserving umbilical cord tissue according to claim 1, wherein the step (a) further comprises: applying a tension to the umbilical cord segment using a tool or a probe.
  6. The method for cryopreserving umbilical cord tissue as described in claim 1, wherein the step (a) further comprises: Insert a tool or a probe into one of the umbilical vessels.
  7. The method of cryopreserving umbilical cord tissue according to claim 6, wherein the tool or the other end of the probe is fixed or pivoted to a fixture.
  8. The method of cryopreserving umbilical cord tissue according to claim 1, wherein the strip of umbilical cord tissue is not treated with an enzyme.
  9. The method of cryopreserving umbilical cord tissue according to claim 1, wherein the step (c) further comprises shaking the strip of umbilical cord tissue in the low temperature composition for 20 to 40 minutes.
  10. The method of cryopreserving umbilical cord tissue according to claim 1, wherein the Watson's gel in the strip-shaped umbilical cord tissue is cut downward.
  11. A method of cryopreserving umbilical cord tissue according to claim 1, wherein the method comprises chopping the strip of umbilical cord tissue into umbilical tissue fragments having a size of less than 0.5 cm per piece.
  12. A method for obtaining umbilical cord tissue-derived stem cells, comprising the steps of: (a) thawing a cryopreserved strip umbilical cord tissue obtained by the method of claim 1, wherein the cryopreserved strip umbilical cord tissue The invention comprises a umbilical cord tissue and a low temperature composition, the strip umbilical cord tissue comprising an umbilical cord blood vessel and a Wittton gel, the Wadden gel comprising a peripheral region of a blood vessel, an intervascular region, and an amniotic membrane a gelatinous structure of the lower region; (b) removing the cryogenic composition; (c) cutting the strip of umbilical cord tissue to form a plurality of umbilical tissue fragments, wherein the umbilical tissue fragments are larger than 2 mm; and (d) The umbilical cord tissue fragments are cultured in a medium to obtain the umbilical cord tissue-derived stem cells.
  13. A method for obtaining umbilical cord tissue-derived stem cells according to claim 12, wherein the umbilical cord The size of the organized fragments is greater than or equal to 0.5 cm.
  14. A method of obtaining umbilical cord tissue-derived stem cells according to claim 12, wherein the method comprises treating the strip of umbilical cord tissue with an enzyme.
TW105104858A 2016-02-19 2016-02-19 Cryopreservation of umbilical cord tissue strips for cord tissue-derived stem cells TWI586804B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200613556A (en) * 2004-08-16 2006-05-01 Cellres Corp Pte Ltd Isolation, cultivation and uses of stem/progenitor cells
WO2008021391A1 (en) * 2006-08-15 2008-02-21 Anthrogenesis Corporation Umbilical cord biomaterial for medical use
CN102686722A (en) * 2009-09-23 2012-09-19 达芬奇生物科技有限责任公司 Umbilical cord lining stem cells and methods and material for isolating and culturing same
TW201311145A (en) * 2011-09-01 2013-03-16 Healthbanks Biotech Co Ltd Storage, cultivation and application of umbilical cord tissue and derived cell thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200613556A (en) * 2004-08-16 2006-05-01 Cellres Corp Pte Ltd Isolation, cultivation and uses of stem/progenitor cells
WO2008021391A1 (en) * 2006-08-15 2008-02-21 Anthrogenesis Corporation Umbilical cord biomaterial for medical use
CN102686722A (en) * 2009-09-23 2012-09-19 达芬奇生物科技有限责任公司 Umbilical cord lining stem cells and methods and material for isolating and culturing same
TW201311145A (en) * 2011-09-01 2013-03-16 Healthbanks Biotech Co Ltd Storage, cultivation and application of umbilical cord tissue and derived cell thereof

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