KR20060032953A - Cryopreservation of human blastocyst-derived stem cells by use of a closed straw vitrification method - Google Patents

Abstract

An improved method for vitrification of biological cells, especially blastocyst-derived stem cells (BS cells). The method is very mild for the cells that remain viable after they have been thawed. The method comprises, i) transfer of the cells to a first solution (solution A), ii) optionally incubation of the cells in the first solution, iii) transfer the cells obtained in step i) or ii) to a second solution (solution B), iv) optionally incubation of the cells in the second solution, v) transfer of the cells obtained from step iii) or iv) into one or more closed straws with dimensions that allow a volume of at least 20 mul to be contained in them vi) sealing the one or more closed straws, and vii) vitrification of the one or more closed straws. An important feature of the present invention is the use of closed straw and that relatively large volumes can be efficiently vitrified and subsequently thawed.

Description

CRYOPRESERVATION OF HUMAN BLASTOCYST-DERIVED STEM CELLS BY USE OF A CLOSED STRAW VITRIFICATION METHOD}

The present invention relates to an improved method of vitrification of biological cells, in particular stem cells (BS cells) derived from blastocysts. This method is a very low irritation method for cells that are still viable after thawing.

Stem cells are cell types that have the unique ability to regenerate themselves and give rise to evolved or differentiated cells. Most cells in the body, such as heart cells or skin cells, are dedicated to performing a particular function, but stem cells are not entwined until they are signaled to develop into differentiated cell types. What makes the stem cells unique is their proliferative capacity as well as the ability to differentiate. For years, researchers have focused on finding ways to use stem cells to replace damaged or dead cells and tissues. To date, most studies have focused on two types of stem cells: embryonic and somatic stem cells. Embryonic stem cells are derived from preimplanted fertilized oocytes, ie blastocysts, while somatic stem cells are present in mature organisms, for example in the bone marrow, epidermis and intestine. According to a number of national laws of Europe and other countries, fertilized oocytes are not recognized as embryos before implantation in the uterus, ie 10-14 days after fertilization, and thus the present invention when such cells are used according to the present invention. In these, it is referred to as stem cells or hBS cells derived from blastocysts. Pluripotency tests demonstrated that the embryo or blastocyst-derived stem cells can give rise to all cells in an organism, including embryonic cells, while somatic stem cells have a more limited repertoire to the cell types to which they are delivered.

In 1998, researchers were able to first isolate embryonic stem cells from human fertilized oocytes and propagate them in culture (see, eg, US Pat. No. 5,843,780 and US Pat. No. 6,200,806).

Increasing research and development within the technical scope of the stem cells requires the use of methods suitable for the conservation of the cells and cell lines. Cells can be vitrified or frozen and stored. Cryopreservation using conventional approaches is very difficult to apply to complex and sensitive biological materials because the extracellular ice formation has a destructive effect. The vitrification method rapidly cools the sample containing the cells to a very low temperature so that the contained water forms a glassy state without crystallization. Thus, supermagnetization is rapid cooling of the liquid medium without ice crystal formation. Amorphous glass is formed upon rapid cooling, for example by direct yielding of the straw containing the cells into liquid nitrogen. The glass maintains a normal liquid distribution but remains in supercooled form. The glass is free of ice crystals and does not cause cell damage to the cells, which may be associated with ice crystal formation. Thus, supermagnetization is defined as the condensation of the amorphous glassy state in which ice nucleation and growth has been prevented in advance.

Cryopreservation of human embryonic stem cells has been studied, and Reubinoff et al. (Human Reproduction, 2001, 16, 2187-2194) described a cryopreservation method of the cells by using a straw-stretched vitrification method. Started. The drawback of the method is that the method involves contacting the open end of the straw with liquid nitrogen, which may be a contaminant of the biological material to be magnetized. Furthermore, due to the dimensions of the pulled straw, the volume of vitrified by Roibinov et al. Was approximately 1 μl.

A method of vitrification that avoids direct contact with nitrogen is described in Rabbit-Bejar et al., Theriogenology, 2002, 58: 1541-52, and in mouse oocytes by Chen et al., Human Reproduction. , 2001, 16 (11): 2350-56. These methods all use closed straws pulled in the same manner as known pulled open straws and therefore have the same dimensions as straws used by Roibinov et al. Thus, in both of these methods about 1 to 2 μl volumes are vitrified in each straw.

Slow freeze and quick thaw methods were used for cryopreservation of cell lines. The method is suitable for use for cryopreservation of mouse embryonic stem cells, for example, but the survival of undifferentiated human embryonic stem cells is very poor and most of the cells appear to differentiate or die. Typically, larger volumes of cells were vitrified by such slow freezing methods and recovered less (Reubinoff et al.).

Thus, there is still a need to develop an effective method of vitrification that is easy to handle and involves as few steps as possible, while at least there is no risk of unnecessary contamination of the cells during the process. In particular, there is a need to develop effective vitrification methods for larger volumes of cells or cell lines, such as hBS cells or hBS cell lines.

Figure 1 shows the thawing recovery rate after demagnetization and demagnetization of human BS cell line SA001.

Figure 2 shows the thaw recovery after demagnetization and demagnetization of human BS cell line SA002.

Figure 3 shows the thaw recovery after demagnetization and demagnetization of human BS cell line AS034.

4 (A)-(C) show typical morphology of human BS cells SA001 cultured on mouse embryonic feeder cells just prior to vitrification.

5 (A)-(C) show the typical morphology of human BS cell SA001 cultured on mouse embryonic feeder cells in the first passage culture after devitrification.

FIG. 6 shows a typical morphology of human BS cell SA001 after desupersonation cultured on mouse embryo feeder cells at 18 (A), 23 (B), 29 (C) and 35 (D) passages.

7 shows (A) human BS cell colonies [p19], (B) SSEA-1 [p31], (C) SSEA-3 [p31], (D) SSEA-4 [p31], (E) TRA-1 -60 [p31], (F) TRA-1-81 [p31], (G) Oct-4 [p31], and (H) ALP [p31].

8 shows karyotype of cell line SA001 after vitrification.

Figure 9 shows in vitro differentiation of desuperstitial human BS cells (SA001) at 29 passages. (A) β-III-tubulin, (B) desmine, (C) α-fetoprotein, (D) HNF-3β.

10 shows in vivo differentiation of human BS cells (SA001) at 19 passages. (A) endoderm (secretory epithelium), (B) mesoderm (cartilage), (C) ectoderm (neural ectoderm).

11 shows a syringe with a closed straw prepared for freezing.

The present invention relates to an improved method of vitrification of biological cells, in particular stem cells (BS cells) derived from blastocysts.

As mentioned above, since the establishment of human blastocyst stem cell banks, there is a need for efficient cryopreservation methods for the development and use of stem cells derived from blastocysts. Effective freezing and thawing techniques allow for the efficient conservation of cells and cell lines. For some purposes, it may be desirable to vitrify large amounts in each straw. This is the case, for example, when a large number of cells are needed in a given process (or use) or when the cells have to be dispatched by mail in an initialized state.

The present invention

i) transfer the cells to the first solution (solution A),

ii) optionally culturing cells in said first solution,

iii) transfer the cells obtained in step i) or ii) to a second solution (solution B),

iv) optionally culturing cells in said second solution,

v) transfer the cells obtained in step iii) or iv) to one or more closed straws having dimensions such that they can contain a volume of at least 20 μl,

vi) sealing said at least one closed straw,

vii) vitrify the one or more closed straws

It relates to a method of vitrification of cells, including.

A very important feature of the above mentioned method is the large volume that can be magnetized in each straw. The present invention provides about 20 μl to about 250 μl, such as about 20 μl to about 225 μl, about 25 μl to about 200 μl, about 25 μl to about 175 μl, about 25 μl to about 150 μl, about 30 μl to A method of vitrifying cells in a closed straw with dimensions that can contain a volume of about 125 μl, about 30 μl to about 100 μl, about 35 μl to about 75 μl, about 40 μl to about 50 μl. will be. The straw used in the examples provided herein has a length of approximately 13 cm, a diameter of about 2 mm and a very thin plastic wall of about 0.1 mm (closed straw, French mini-stro, 250 μl, L 'Aigle, IMV ZA 475 °, 133 mm, Svensk Mjolk). However, if the diameter and thickness of the straw is approximately the same as the straw used in the present invention (the dimensions of the container with liquid nitrogen is such that the entire length of the straw can be covered with liquid nitrogen), a longer straw is used. It can be expected that even more volumes can be successfully vitrified.

Another very important step in the above mentioned method is the use of so-called closed straws. In the present text, the term "closed straw" has an open end at the filling position, allowing for example to be filled with a biological material (e.g. a cell or cell line) in a suitable medium, but the end is firmly immediately after filling It is used to represent a straw that is closed to prevent unnecessary contamination from the periphery of the cell and also to prevent the risk of unnecessary contamination from the cell from the periphery. Airtight sealing at both ends of the straw is important to prevent contamination of both the sample and the surroundings. A suitable system is a passive sealing unit called CBS Cryo Bio System (SYMS).

It is important that the straw is open on one side and plugged on the other. The stopper is capable of inhaling air with a syringe to fill the straw with liquid, but once in direct contact with the liquid it polymerizes to seal the capillary at the end. Other suitable sealing methods of the ends can also be applied. The other end will then be closed using a seal (welding, bond, etc.). What is important is the small diameter and thin wall to allow quick freezing of the contents in the straw. The length is not critical but for practical reasons it is advisable to have a standard length to fit the standard holder in the liquid nitrogen tank. The straw is made of plastic but may be made of any suitable material, for example glass (which may be more fragile). Importantly, the material is safe and no material can be absorbed or released, i.e. non-porous, non-toxic and biocompatible.

In the present application, the term "freezing preservation" refers to the preservation of biological material at cryogenic temperatures.

As used herein, the term "directly contacted" or "directly exposed" means that the biological material is brought into contact with the frozen material, for example the surface of the biological material, or the medium, solution or material in which the material is present. "Directly contacted" or "directly exposed" to the frozen material.

As used herein, the term "freeze substance" refers to any substance that can cause the vitrification of a biological substance. In theory, any freezing medium that is sufficiently cold can be used because the sample is not in direct contact with the freezing medium. Suitable materials include, but are not limited to, liquid gases such as liquid nitrogen, liquid propane, liquid helium, ethane and the like.

As used herein, "growable" means that a biological material can survive, develop, and multiply normally for a period of time.

According to the present invention, about 20 μl of volume of biological material (eg hBS cells or hBS cell lines) is placed in a closed straw. The closed straw is then exposed to frozen material (suitably liquid nitrogen). Upon exposure to the frozen material, the cells undergo vitrification and can then be stored for some time and thawed later. The thawed biological material still remains viable. As is apparent from the above, there is no direct exposure or direct contact of the cells with the frozen material. Thus, there is no risk of contamination of the cells (from external sources) as well as contamination of the environment by the cells.

The biomaterials of the present invention are living cells or cell lines, in particular cells derived from BS cells, BS cells or BS cells. The cell may be at any stage of development. Preferably, the cell is a mammalian source, such as but not limited to human, non-human primates such as monkeys, laboratory animals such as rats, mice and hamsters, agricultural livestock such as pigs, sheep Obtained from animal sources, including cattle, goats and horses. In a particularly interesting embodiment of the invention, said cell is a human stem cell, including a human BS cell.

Suitable cells for use in the method according to the invention are BS cells or BS cell lines, in particular hBS cells or hBS cell lines. Such cells or cell lines can be obtained using the procedures disclosed herein.

One or more of the supermagnetization solutions (first and second solutions) may contain one or more cryoprotectants or mixtures of cryoprotectants. Non-toxic cryoprotectants are of course preferred. Cryoprotectants help minimize shrinkage by reducing the mole fraction of other solutes remaining in unfrozen water. This inhibits the formation of crystalline ice, thus lowering the freezing point of water. This can also prevent protein denaturation by hydrogen bonding with the bond water. As the cells cool, the solvent water converts to extracellular ice, and increasing extracellular concentrations of impermeable electrolytes or non-electrolytes damage the cells. When treated with a cryoprotectant, the cells do not reach the salt concentration of the untreated cells until they reach much lower temperatures. The chemical reaction proceeds very slowly at these low temperatures and consequently minimizes cell damage. Usually it is better to use a combination of cryoprotectants because there may be differences between the different types. The cryoprotectants may also act as osmotic actives. Suitable cryoprotectants can be selected from the group consisting of ethylene glycol, propylene glycol, dimethylsulfoxide, glycerol, propane diols, sugars such as sucrose, trehalose, maltose, lactose and methyl pentane diol. The concentration of the individual agents contained in the first and / or second solution is typically 5 to 50% v / v, for example about 5% to about 40% v / v, for example about 5% to about 25 % v / v (first solution) and about 5% to about 30% v / v (second solution). Typically, the total concentration of cryoprotectant in the second solution (calculated as v / v, w / v or M) is greater than the concentration in the first solution. The first and second solutions may contain the same or different one or more cryoprotectants. The concentration of one or more cryoprotectants in the first and second solutions may be the same or different, typically the total concentration of the cryoprotectant in the second solution is greater than the concentration in the first solution.

In a particular embodiment of the invention, the cryoprotectant is trehalose. The concentration of trehalose contained in the first and / or second solution is typically about 0.02 M to about 1 M, for example about 0.05 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.2 M to about 0.7 M, about 0.3 M to about 0.65 M, about 0.4 M to about 0.6 M, about 0.45 M to about 0.55 M. Usually, sucrose is used for similar purposes. Trehalose is a unique natural disaccharide and is found in hundreds of plants and animals. Trehalose is an important energy source and has proven to be a major factor in the stabilization of organisms during the freezing period. Trehalose has been shown to lower the phase transition temperature of the cell membrane, allowing the cell membrane to remain in the liquid crystal state even when dried. While not wishing to be bound by any theory, it is assumed that it prevents cell membrane leakage during rehydration, thus preserving cell viability. Regarding the protein, trehalose has been shown to inhibit protein denaturation by the exclusion of water from the protein surface when the cells are in a hydrated state.

In another embodiment of the invention, the cryoprotectant is sucrose. The concentration of sucrose contained in the first and / or second solution is typically about 0.02 M to about 1 M, for example about 0.05 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.2 M To about 0.7 M, about 0.3 M to about 0.65 M, about 0.4 M to about 0.6 M, about 0.45 M to about 0.55 M.

In yet another embodiment of the present invention, at least one of the first and second solutions comprises a cryoprotectant.

At least one of the first and second solutions may comprise a viscosity modifier. Suitable viscosity modifiers for use in the present disclosure can be selected from the group consisting of Ficoll, Percoll, hyaluronic acid, albumin, polyvinyl pyrrolidone, alginic acid, gelatin and glycerol. The first and second solutions may contain one or more viscosity modifiers, the same or different. The concentration of one or more viscosity modifiers in the first and second solutions may be the same or different.

In a particular embodiment of the invention, the viscosity modifier is picol. The concentration of picol contained in the first and / or second solution is about 150 mg / ml or less, for example about 100 mg / ml or less, about 50 mg / ml or less, about 25 mg / ml or less, about 15 mg Or less than or about 10 mg / ml.

In one embodiment of the present invention, at least one of the first and second solutions is an aqueous solution.

In a particular embodiment of the invention, step ii) of the aforementioned method is included.

The possible duration can be from 10 seconds to 20 minutes, because at this stage the point of time enhances equilibrium with the solution and ensures that the cryoprotectant is adequately sprayed, but in the presence of DMSO, the cells are too toxic to DMSO. It should not be exposed for a long time.

The culture is typically for a period of 5 seconds to about 20 minutes, for example about 10 seconds to about 15 minutes, about 15 seconds to about 10 minutes, about 20 seconds to about 7.5 minutes, about 30 seconds to about 5 minutes, And at about 37 ° C. for a period of about 40 seconds to about 4 minutes, about 50 seconds to about 3 minutes, about 30 seconds to about 2 minutes, about 45 seconds to about 1.5 minutes, or about 1 minute.

In a further embodiment, step iv) is also included and the culture is typically carried out for a period of about 5 seconds to about 10 minutes, for example from about 10 seconds to about 7.5 minutes, from about 10 seconds to about 5 minutes, about 15 seconds to about 4 minutes, about 15 seconds to about 3 minutes, about 15 seconds to about 2 minutes, about 20 seconds to about 1 minute, about 5 seconds to about 1 minute, about 5 seconds to about 30 seconds or about 10 seconds At about 37 ° C. for a period from about 30 seconds.

In a particular embodiment, step iv) is included and the culturing is performed at about 37 ° C. for up to about 30 seconds.

The vitrification method is very efficient and gentle to the cells. Typically, at least about 50% of the cells, for example at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, about 90 At least%, or at least about 95%, are viable after demyocyteization and incubation in a suitable medium. Suitable dedevitrification procedures are disclosed herein.

In another embodiment, the invention also relates to a cell that has undergone vitrification by the method according to the invention.

In a further or separate aspect of the present invention, the present invention relates to a de-supermagnetization method.

The demagnetization is

viii) subjecting the at least one vitrified closed straw to an environment having a temperature from about room temperature to about 40 ° C. during the time that the contents of the closed straw are thawed,

ix) opening said at least one closed straw,

x) subjecting the cells contained in the one or more open straws to a washing process using a third solution (solution C),

xi) optionally transfer the washed cells obtained from step x) to a fourth solution (solution D),

xii) optionally culturing cells in said fourth solution,

xiii) optionally seeding the cells from xii) from the fourth solution and seeding the cells onto feeder cells,

xiv) optionally further culturing the cells

It includes.

The cells obtained after step x) can be used directly for whatever purpose they are desired and any of the above steps can be applied to further examine the cells, for example for viability.

Step viii) relates to thawing of the cells. This point in the present invention merely thaws the contents of the straw and should be done during visual inspection of the straw, so timing is less important. The temperature should not exceed 40 ° C but can be from room temperature to 40 ° C. Higher temperatures can cause heat shock upon thawing of cells unless they are quickly removed from the water bath after thawing.

Thus, the method may also include, as essential steps, steps xi), xiii) and xiv); Moreover it may comprise step xii).

The desupermagnetization solution may contain a cryoprotectant, for example a cryoprotectant with an osmotic activity, for example a low toxicity osmotic active agent, generally avoiding, for example, DMSO and optionally for example trehalose and Sucrose is used alone or together. The high percentage of the disaccharide in the solution prevents cell destruction that may occur by sudden contact with a solution free of DMSO. The presence of the disaccharide outside the cell will prevent natural osmotic forces from functioning and allow enough time for the cell to discard DMSO (or similar) present inside the cell and instead slowly replace water.

Thus, the third and / or fourth solution, where appropriate, typically comprises one or more cryoprotectants.

In certain embodiments, the one or more cryoprotectants are selected from the group consisting of glycerol, trehalose, sucrose, ethylene glycol, DMSO, propanediol and / or mixtures thereof, in particular glycerol, trehalose, sucrose or Mixtures of these are suitable for use.

The concentration of cryoprotectant in the third and / or fourth solution is typically from about 0.02 M to about 1 M, for example from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.15 M to about 0.5 M, about 0.2 M to about 0.4 M, wherein the concentration of the cryoprotectant in the third solution is the concentration of the osmotic active agent in the fourth solution, if appropriate Greater than Typically, the concentration of cryoprotectant in the third solution is greater than the concentration of cryoprotectant in the fourth solution (where appropriate).

The following provides a general disclosure of the method of the invention.

Colonies of human blastocyst derived stem (hBS) cells are cut into pieces (0.1-0.4 mm × 0.1-0.4 mm). Up to 20 (preferably 10) cell pieces in a volume of 40-50 μl can be frozen in a closed straw. Closed straws have plugs on one side and open on the other. After the cell pieces are sucked into the straw for freezing, the ends of the plugs are sealed using cold-PBS, while the open ends are bonded (welded) and heat-sealed (Demtek, A / S) Seal using. A round of test freezing and thawing is performed before freezing more cells. After thawing, the cell pieces are seeded in a culture dish with mouse embryonic feeder cells (MEF). Human BS cells are passaged once and subsequently evaluated.

All percentages mentioned are v / v.

The following description provides instructions for suitable procedures, solutions, durations, and the like. However, based on the general description and guidelines of the present invention, those skilled in the art can change different elements within the scope of the present invention.

Supermagnetization  Course-General Description

Ready:

1. Prepare a mother liquor consisting of 0.6 M trehalose in cold-PBS obtained from Vitrolife AB (Gothenburg, Sweden).

2. Solution A:

10% ethylene glycol is prepared in cold-BPS and sterile filtered.

Sterile DMSO is added to a final concentration of 10%.

3. Solution B:

A solution consisting of 0.3 M trehalose and 20% ethylene glycol is prepared in cold-PBS and sterile filtered.

Sterile DMSO is added to a final concentration of 20%.

4. Place 1 ml of Solution A and Solution B, respectively, in two separate wells in a 4-well plate (Nunclon, VWR International). Place the plate at 37 ° C.

5. Selected colonies of stem cells derived from human blastocysts showing suitable morphology when cut for passage culture using autoclaved pulled glass capillary (World Precision Instruments) (or stem cell cutting tool from Swemed). Cut in the same manner as The cutting tool from Swemed is a sharp sterile glass capillary, has an angle of 25 degrees and 200 or 300 micrometer lumens and is designed for cutting, manipulating and delivering hBS colonies, or portions of hBS colonies. It is produced by Swemed Lab International AB, Billdal, Sweden.

6. Attach the hBS-number to the required number of straws (closed straw, French mini-stro, working volume 250 μl, L'Aigle, IMV ZA 475 °, 133 mm, Svensk Mjolk) and freeze code on the visiotube. Attach a table (ie cell line / date / signature).

Freezing process:

1. Transfer the cells to be frozen in the straw into Solution A using a glass capillary (World Precision Instruments) or a pulled glass pipette (Pasteur, VWR International).

2. Incubate the cells in solution A for 1 minute.

3. The cells are then transferred to one drop (25 μl) of Solution B, and within 25 seconds the cells are transferred to another new drop of Solution B (25 μl).

4. Incubate the cells in Solution B for 25 seconds (maximum). The desired time for 3-4 should be as short as possible.

5. Connect 1-1.5 cm of tubing (autoclaved) to a 1 ml syringe (Tuberculin syringe, disposable, Codan Triplus AB), which in turn is connected to the end (closed end) of the cotton yarn stopper of the straw. Let's do it. The silicone tubing acts as a seal between the straw and the syringe. First, cold-PBS is sucked into the straw into an approximately 2-3 cm column. Subsequently, approximately 1 to 2 cm of air is sucked in (see FIG. 1).

6. The cells are then drawn into solution from the solution B on a 2 cm column under a stereo microscope.

7. Inhale approximately 1 to 2 cm of air followed by 0.5 to 1 cm of cold-PBS (which acts as an extra plug at the end).

8. The contents of the straw are sucked into a syringe such that the cold-PBS contacts the cotton yarn stopper (which swells the stopper).

9. Remove the straw from the syringe using a pair of tweezers and then seal it by welding using a heat seal device.

10. Place the straws in the geotubes, which in turn are placed in a tank containing liquid nitrogen for long term storage.

mask Supermagnetization  Course-General Course

Ready:

1. Prepare a mother liquor consisting of 0.2 M trehalose in cold-PBS.

2. Solution C: Sterile filtration of 0.2 M trehalose in cold-PBS.

3. Solution D: 0.1 M trehalose in cold-PBS is prepared and sterile filtered.

4. Add 1 ml of Solution C, Solution D and hBS-medium (see below) to individual wells in 4-well plates (Nunclon, VWR International) and incubate the plates at 37 ° C.

[The hBS medium is 20% KNOCKOUT® serum supplement, and the following components at each final concentration: penicillin 100 units / ml, non-essential amino acids 0.1 mM, L-glutamine 2 mM, β-mercapto 100 μM of ethanol, 4 ng / mL of human recombinant bFGF (basic fibroblast growth factor), KNOCKOUT Dulbecco's Modul Eagle's Medium.

It is important to quickly wash the thawed cells from DMSO used in the vitrification solution. The presence of less toxic trehalose contributes to a relatively slow stepwise change from the vitrification solution to the medium used for seeding of the cells. The concentration can also be varied at different efficiencies (5-50% v / v, w / w or w / v). It is also possible to use other cryoprotectants of low toxicity.

thaw

1. Closed straw containing vitrified human BS cells is collected from a storage tank containing liquid nitrogen and placed in a vial containing liquid nitrogen.

2. The closed straw is kept in air for 10 seconds at room temperature and then placed in a water bath at 40 ° C. for about 2 seconds. The straw is dried using autoclaved “allduk”.

3. Use the autoclaved scissors to cut open only the side of the seal at the blocked end of the straw. The straw is attached onto a syringe using a piece of silicone tubing as a seal. The straw is then cut open at the bond (welding). Air in the syringe is used to push hBS cells into solution C. The amount of cells washed in the same well should not exceed the amount contained in one single straw.

4. Incubate the hBS cell colonies in solution C for 1 min. Usually, it is incubated for about 1 minute to about 20 minutes.

5. Under stereomicroscopy, transfer the hBS cell colonies into solution D using glass capillary (World Precision Instruments) or a pulled glass pipette (Pasteur, VWR International).

6. Incubate the hBS cell colonies in solution D for 5 minutes. Usually about 5 minutes to about 30 minutes incubation.

7. Under stereomicroscopy, the hBS cell colonies are transferred to hBS-medium using a glass capillary (World Precision Instruments) or a pulled glass pipette (Pasteur, VWR International).

8. Transfer the colonies from the hBS-medium in a few seconds (> 5 seconds) using a glass capillary (World Precision Instruments) or a pulled glass pipette (Pasteur, VWR International) and in a culture dish mouse embryonic feeder cells (MEF) Seeding at the top of the

9. Place the petri dish into the incubator for further incubation.

The invention is further illustrated below with the following examples, which are not intended to limit the invention in any way.

Example  One

hBS cells Supermagnetization

Two solutions A and B were prepared (solution A: sterile filtered 10% ethylene glycol in cold-PBS, 10% DMSO; solution B: sterile filtered 0.3 M trehalose in cold-PBS, 20% ethylene glycol, 10% DMSO). Selected colonies of stem cells derived from human blastocysts that exhibit a suitable morphology are cut in the same manner as when the cells are cut for normal passage culture using a stem cell cutting tool (Swede Labs International, Billdal, Sweden). . The cell pieces should be about 0.1 to 0.4 mm x 0.1 to 0.4 mm in size. The cell pieces are first incubated in 500 μl of preheated (37 ° C.) Solution A for 1 minute, then transferred to 25 μl Solution B, incubated for 30 seconds and then transferred to a new drop of Solution B and incubated for 30 seconds. The volume is about 40-50 μl. About 10 cell pieces are sucked into a straw prepared for vitrification and then the straw is closed with a bond. The straw is immersed in liquid nitrogen.

Example  2

Supermagnetized  Thawing of Human BS Cells

Two solutions C and D are prepared (solution C: sterile filtered 0.2 M trehalose in cold-PBS; solution D: sterile filtered 0.1 M trehalose in cold-PBS). Solutions C and D and hBS-medium are preheated at 37 ° C. Closed straw containing vitrified hBS cells (about 10 cell pieces) is removed from the liquid nitrogen tank. The straw is stored at room temperature for 10 seconds and then thawed rapidly in a 40 ° C. water bath (within seconds). The stray ends are cut open using autoclaved scissors and the contents are pushed from the straw into solution C using a syringe. hBS cells are incubated for 1 minute in 500 μl Solution C, transferred to 500 μl Solution D and incubated for 5 minutes. HBS cell pieces are rapidly washed under hBS medium under a stereo microscope and then seeded on top of mouse embryo feeder cells in hBS medium in a culture dish. The cells are then cultured (cultivated at 37 ° C.) and numbered new colonies established and passaged to confirm viability of the hBS cells after vitrification. The recovery rate of viable cells following the vitrification and thawing process is usually in the range of 70 to 100%.

Example  3

Of human BS cells using closed straw Supermagnetization  And thawing

Human BS cells (cell lines SA002, SA121 and SA181) were vitrified and thawed according to the procedure described in Examples 1 and 2. The hBS cell colonies were evaluated and counted 48 hours after seeding on top of mouse embryonic feeder cells in a culture dish. Thawing recovery was calculated as the ratio between the number of viable thawed colonies (indicating the appropriate hBS cell morphology) and the number of original vitrified hBS cells (since each of these cell pieces could produce one colony). ). Three straws per cell line were prepared and evaluated and the results are provided below for each individual straw, indicating a recovery of 40% to 100%.

     Closed straw                   Human BS cell line           SA002            SA181      Thawing recovery rate            3/7             9/9           5/10             6/8          10/10             2/5

Example  4

Direct comparison between closed and pulled open straws

Human BS cells (cell line AS034) were vitrified and thawed according to the procedures described in Examples 1 and 2, except that pulled and opened straws were used in parallel with closed straws. Remarkably, only about 4 BS cell pieces can be vitrified in each pulled open straw. The hBS cell colonies were evaluated and counted 48 hours after seeding on top of mouse embryonic feeder cells in a culture dish. Thawing recovery was calculated as the ratio between the number of viable thawed colonies (showing the appropriate hBS cell morphology) and the number of original vitrified hBS cells. Three straws per cell line were prepared and evaluated and the results are provided below for each individual straw, which yields more viable cells (absolute number) from each of these closed straws while maintaining an acceptable recovery rate. Indicates that it can.

                Human BS Cell Line AS034       Closed straw  Pulled open straw       Thawing recovery rate            6/9             4/4            6/9             2/4           6/10             3/4

Example  5

Supermagnetization  On the badge Trehalozwa Sucrose  Comparison between what you use

Human BS cells (cell line SA121) can be prepared according to the procedures described in Examples 1 and 2, or by using trehalose in the vitrification and desupermagnetization medium and at the same molar concentration as that used for the trehalose. Ultrasonization and thawing were followed according to the procedure described in Examples 1 and 2 except using sucrose. The hBS cell colonies were evaluated and counted 48 hours after seeding on top of mouse embryonic feeder cells in a culture dish. Thawing recovery was calculated as the ratio between the number of viable thawed colonies (showing the appropriate hBS cell morphology) and the number of original vitrified hBS cells. Three straws per cell line were prepared and evaluated and the results are provided below for each individual straw, indicating that there is no significant difference between using trehalose and sucrose.

Human BS Cell Line SA121 Trehalose Sucrose Thawing recovery rate 5/9 8/10 8/10 5/10 6/10 9/10

Example  6

Supermagnetization  In the badge Picol  Use of human BS cells Supermagnetization  And thawing

Human BS cells (cell line SA121) were vitrified and thawed according to the procedure described in Examples 1 and 2 except using Picol (0 mg / ml, 10 mg / ml and 100 mg / ml) in vitrification medium. I was. The hBS cell colonies were evaluated and counted 48 hours after seeding on top of mouse embryonic feeder cells in a culture dish. Thawing recovery was calculated as the ratio between the number of viable thawed colonies (showing the appropriate hBS cell morphology) and the number of original vitrified hBS cells. Three straws per cell line were prepared and evaluated and the results are provided below for each individual straw.

Closed straw Human BS Cell Line SA121 Picol concentration 0 mg / ml 10 mg / ml 100 mg / ml Thawing recovery rate 5/5 7/10 6/10 9/9 6/10 3/10 6/10 3/10 -

Example  7

Supermagnetization  Different concentrations in the medium Trehalose  Comparison between things you use

Human BS cells (cell line SA121) according to the procedure described in Examples 1 and 2 except that trehalose was used at two different concentrations (0.3 M and 0.5 M) in the second vitrification solution (solution B) Supermagnetize and thaw. When 0.3 M trehalose was used for solution B, solution C contained 0.2 M trehalose and solution D contained 0.1 M trehalose. When 0.5 M trehalose was used for solution B, solution C contained 0.4 M trehalose and solution D contained 0.2 M trehalose. The hBS cell colonies were evaluated and counted 48 hours after seeding on top of mouse embryonic feeder cells in a culture dish. Thawing recovery was calculated as the ratio between the number of viable thawed colonies (showing the appropriate hBS cell morphology) and the number of original vitrified hBS cells. Two separate experiments were performed using two different human BS cell lines. Three straws per cell line were prepared and evaluated and the results are provided below for each individual straw. The results show that 0.5 M trehalose in solution B works better than 0.3 M trehalose in solution B, but all of the investigated trehalose conditions work well.

Closed straw Human BS Cell Lines SA121 and SA002 Trehalose 0.3 M 0.5 M Thawing recovery rate (SA121) 6/10 8/10 5/10 9/10 6/10 8/8 Thawing recovery rate (SA002) 5/8 7/8 6/8 8/8 7/7 5/7

Example  8

Closed straw Supermagnetization  And ride Supermagnetization  Extensive assessment of the process

To assess the quality of the vitrification method, large amounts of human BS cells were vitrified in three different cases using three different human BS cell lines (SA001, SA002 and AS034) (performed as described in Example 1 above). ). In each case, straws of> 100 were vitrified from each cell line. Eight to ten straws from each of these large batches were randomly selected, demystified (performed as described in Example 2 above) and seeded on top of mouse embryonic feeder cells in separate dishes. In each dish, the number of hBS cell masses that were seeded, adhered, expanded and displayed the appropriate morphology was measured. The results are shown in FIGS. 1, 2 and 3, which produced hBS cell colonies capable of growing all straw, which were subsequently passaged and characterized according to standard procedures.

Example  9

Supermagnetization  And typical morphology of human BS cells before and after thawing

A typical form of human BS colony (cell line SA001) prior to vitrification is shown in FIG. 4. After desupervitation and seeding, viable colonies proliferated and exhibited a characteristic morphology in undifferentiated human BS cells (FIG. 5). Subsequently, these cells were proliferated and passaged according to standard procedures, a typical example of the human BS cell colonies is shown in FIG. 6. Similar results were obtained for human BS cell lines SA002 and AS034 (data not shown).

Example  10

In a closed straw Supermagnetization  And ride A supersonist  Follow-up Characterization of Human BS Cells Affected

In order to demonstrate that human BS cells fully recover after the vitrification and demagnetization process and exhibit suitable characteristics, extensive characterization of the hBS cells was performed. This includes analysis of surface antigen expression, karyotyping, and in vitro as well as in vivo pluripotency testing. The following results were obtained using human BS cell line SA001, and similar results were also obtained using human BS cell line SA002 and AS034 (data not shown).

Immunohistochemical Staining of Undifferentiated hBS Cells

De-minorized human BS cells (cell line SA001) cultured on mouse embryonic feeder (MEF) cells were immobilized in PFA and subsequently made permeable using Triton X-100. After successive washing and blocking steps, the cells were incubated with the primary antibody (as indicated). Conjugated secondary antibodies were then used for detection. Nuclei were visualized by DAPI staining. The activity of alkaline phosphatase (ALP) was measured using a commercially available kit according to the manufacturer's instructions (Sigma Diagnostics, Stockholm, Sweden). Passage culture numbers on which each analysis was performed are shown in parentheses in the figure legend for FIG. 7. As illustrated in FIG. 7, the results indicated that human BS cells showed positive staining for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4 and ALP and were not differentiated. Negative for SSEA-1 as expected for human BS cells.

Karyotyping

De-minorized human BS cells (main SA001) cultured on MEF were cultured in the presence of Calicurin A and then washed with cell culture medium. The cells were harvested by centrifugation and fixed using ethanol and glacial acetic acid. Chromosomes were visualized using trypsin-Giemsa staining. As illustrated in FIG. 8, the results show that there is no detectable chromosomal abnormality in the cells following the method of vitrification and demagnetization.

Telomerase  activation

To analyze telomerase activity, the Telo TAGGG telomerase PCR ELISA ^PLUS kit (Roche, Basel, Switzerland) was used according to the manufacturer's instructions. The assay uses the internal activity of telomerase and amplifies the product by PCR and detects it by enzyme linked immunosorbent assay (ELISA). Human BS cell line SA001 was analyzed after incubation and culture on mouse embryonic feeder cells and showed high telomerase activity. High telomerase activity in hBS cells correlates with the cell's ability to divide indefinitely in culture.

In vitro differentiation

To examine the pluripotency of de-supermagnetized human BS cells, undifferentiated colonies from cell line SA001 were transferred to suspension cultures using stem cell cleavage tools (Swemed Lab, Goteborg, Sweden) to identify embryonic analogues (EBs). Formed. Subsequently, EB was placed on a tissue culture plate and antibodies directed against β-III-tubulin (endoderm), desmin (mesoderm), α-fetoprotein and HNF-3β (endoderm) against spontaneously differentiated cells Immunohistochemical evaluation was performed using the Spontaneously contracting cells resembling cardiomyocytes were also observed (not shown). Together, the results indicate that human BS cells subjected to the vitrification and devitration methods maintain the ability to differentiate into cells exhibiting three different germ layers in vitro (ie, the cells are still pluripotent). The results are illustrated in FIG. 9.

In vivo differentiation

To investigate the pluripotency of de-supermagnetized human BS cells, undifferentiated cells (cell line SA001) were surgically placed under the renal membrane of severe combined immunodeficiency (SCID) mice. The mice were killed after 8 weeks and tumors were excised and fixed in PFA. Tissue evaluation of hematoxylin-eosin stained paraffin sections was performed to determine the presence of tissue derived from all three germ layers. As illustrated in FIG. 10, human BS cells that underwent the method of vitrification and demagnetization maintained the ability to differentiate into cells exhibiting three different germ layers in vivo (ie, the cells are still pluripotent). .

Supermagnetization  General method for the establishment of cells that can be used in the process

The PCT application published on Jul. 10, 2003, ie, WO 03/055992 (same applicant) after the priority date of the present invention, discloses a method for establishing hBS cells suitable for use in the method of the present invention. In one aspect of the invention, the cells used are obtained by the method claimed in WO 03/055992, which is incorporated herein by reference.

The method of establishing stem cells or cell lines derived from pluripotent human blastocysts from fertilized oocytes comprises the following steps:

i) obtaining modified blastocysts, optionally of grade A or B, to obtain blastocysts of grade A or B,

ii) culturing the blastocyst with feeder cells to establish one or more colonies of genus cell mass cells,

iii) isolating said genus cell mass cells by mechanical incision,

iv) culturing the genus cell mass cells with feeder cells to obtain stem cell lines derived from blastocysts,

v) optionally propagating stem cell lines derived from said blastocyst.

As starting material for this process, fertilized oocytes are used. The quality of the fertilized oocyte is important for the quality of the resulting blastocyst. In step i) of the method human blastocysts can be obtained from frozen or fresh human ex vivo fertilized oocytes. The following describes a selection process for oocytes suitable for use in the method according to WO 03/055992. An important success criterion of the method of the invention has been found to be the selection of suitable oocytes. Thus, when only grade 3 oocytes are applied, the probability of obtaining hBS cell lines that meet the general requirements (discussed below) is low.

Donated fresh fertilized oocytes:

On day 0, the oocytes are soaked in Asp-100 (Vitrolife) and on day 1 fertilized in IVF-50 (Vitrolife). The fertilized oocytes are evaluated on the 3rd day based on morphology and cell division. The following grades are used for the evaluation of modified oocytes:

Grade 1 fertilized oocytes: uniform blasts, no fragments

Grade 2 fertilized oocytes: <20% sections

Grade 3 fertilized oocytes:> 20% sections

After evaluation on day 3, grade 1 and grade 2 modified oocytes are implanted or frozen for storage. Grade 3 fertilized oocytes are transferred to ICM-2 (Vitrolife). The fertilized oocytes are further cultured for 3 to 5 days (ie 5 to 7 days after fertilization). The blastocysts are evaluated according to the following rating:

Grade A blastocysts: Expanded with a pronounced genome cell mass (ICM) on day 6

Class B blastocyst: not expanded but the same as class A

Class C blastocyst: ICM not visible

Donated, frozen fertilized oocytes:

On day 2 (after fertilization) the fertilized oocytes are frozen at the 4-cell phase using a free-kit (Vitrolife). Frozen fertilized oocytes are stored in liquid nitrogen. Obtain informed consent from a donor who is five years old. The fertilized oocytes were thawed using a thaw kit (Vitrolife) and the procedure described above was performed on day 2.

As mentioned above, fresh fertilized oocytes are of grade 3 quality and frozen fertilized oocytes are of grade 1 and grade 2. According to the data obtained by this establishment method, the percentage of fresh fertilized oocytes developing into blastocysts is 19%, while 50% of frozen fertilized oocytes develop into blastocysts. This means that the frozen fertilized oocytes are much better at obtaining blastocysts, due to a better-than-expected fertilized oocyte. 11% of blastocysts from fresh fertilized oocytes develop stem cell lines, whereas 15% of blastocysts from frozen fertilized oocytes develop stem cell lines. In summary, of cultured fertilized oocytes, 2% of fresh fertilized oocytes developed stem cells, and 7% of cultured frozen fertilized oocytes developed stem cells.

Culture of the fertilized oocytes into blastocysts is carried out according to procedures well known in the art. The preparation of blastocysts is described in Gardner et al., Embryo culture systems, In Trounson, A.O., and Gardner, D.K. (eds), Handbook of in vitro fertilization, second edition. CRC Press, Boca Raton, pp. 205-264; Gardner et al., Fertil Steril, 74, Suppl 3, O-086; Gardner et al, Hum Reprod, 13, 3434, 3440; Gardner et al, J Reprod Immunol, In press; and Hooper et al, Biol Reprod, 62, Suppl 1, 249.

After establishment of blastocysts from fertilized oocytes optionally of grade 1 or 2 in step i), the blastocysts of grade A or B are cultured with feeder cells for the establishment of one or more colonies of genus cell mass cells. . After plating on feeder cells, monitor for growth and when the colonies are large enough for manual passage culture (approximately 1 to 2 weeks after staining), the cells are excised from other cell types and grown on new feeder cells by growth. Can be extended Isolation of the genus cell mass cells is performed by mechanical excision, which can be done by using glass capillaries as cutting tools. The detection of the genus cell mass cells is readily performed visually under a microscope, and therefore the oocytes do not need to be treated with enzymes and / or antibodies to damage or remove the feeder cell layer.

Thus, the procedure of WO 03/055992 alleviates the need for immunosurgery. By comparing the success rate in the use of immunosurgery with the method of the present invention (leave the nutrient cell layer intact), a much simpler, faster and traumatic immune surgery avoidance procedure has been observed to be more efficient than immunosurgery. This process allows commercial production of stem cell lines and the differentiation of these cell lines. From a total of 122 blastocysts, 19 cell lines were established (15.5%). 42 blastocysts were treated by immunosurgery, 6 of which successfully established cell lines (14%). 80 blastocysts were treated by the method of the present invention and 13 cell lines were established (16%).

Following ablation of the genus cell mass, the genus cell mass cells are incubated with feeder cells to obtain stem (BS) cell lines derived from blastocysts. After obtaining the hBS cell line, the cell line is optionally propagated to expand the amount of cells. Thus, stem cell lines derived from the blastocysts can be propagated by passage culture, for example, every 4 to 5 days. If the stem cell lines are cultured longer than 4-5 days before passaging, the probability of undesirably differentiation of the cells increases.

Specific passage cultures of cells in a trophic culture system are provided in Example 5 herein.

Human BS cell lines can be isolated from spontaneously hatched blastocysts or from expanded blastocysts with a complete clear zone. In the above method, the blastocyst of step i) is a spontaneously hatched blastocyst. For hatched blastocysts, the feeder cell layer may remain intact. Hatched blastocysts or blastocysts with clear or partially cleared zones may be placed on inactivated feeder cells.

Prior to step ii), the clear zone of the blastocyst is treated with, for example, one or more acidic agents, for example ZD ^™ -10 (Vitrolife, Gothenburg, Sweden), one or more enzymes or mixtures of enzymes, for example pronase By at least partially cutting or chemically corrugated.

Simple Sigma treatment on blastocysts with a complete transparent band eliminates the band. Other types of proteases that have the same or similar protease activity as pronase can also be used. The blastocysts can be plated on the inactivated feeder cells following pronase treatment.

In embodiments of the invention step ii) and / or step iv) may be carried out in an agent that improves adhesion of blastocysts and / or genus cell mass cells to feeder cells. Suitable materials for this purpose are hyaluronic acid.

Suitable media for smearing blastocysts on feeder cells may be hBS-medium, which may be supplemented with hyaluronic acid, which appears to promote adhesion of feeder cells to feeder cells and growth of the genus cell mass. Hyaluronan (HA) is an important glycosaminoglycan that constitutes an extracellular matrix in the joint. It appears to exert its biological effects through binding interactions with two or more cell surface receptors, namely CD44 and receptors for HA-mediated motility (RHAMM), and proteins in the extracellular matrix. The positive effect of HA during the establishment of hBS cells can be exerted through interaction with the surfactant polar head of phospholipids in the cell membrane, thereby stabilizing the surfactant layer and thus increasing the surface tension of the genus cell mass or blastocyst. Can be lowered to increase the binding efficiency to feeder cells. On the other hand, HA can exert a biological effect that binds to the genus cell mass or blastocyst and / or its receptor on feeder cells, which positively affects the adhesion and growth of the genus cell mass. Accordingly, other agents may also be used in place of hyaluronic acid, which may alter the surface tension of the fluid or otherwise affect the interaction between the blastocyst and the feeder cells.

In the above-described method, the culture of the feeder cells is important for the establishment of the hBS cell line. Proliferation of blastocyst-derived stem cell lines may comprise passage of the feeder cells up to three times, for example up to two times.

Suitable feeder cells for use in the methods of the invention are, for example, fibroblasts of embryonic or adult origin. The feeder cells used in steps ii) and iv) in the method according to the invention are the same or different and from any mammal, including animal origin, for example humans, mice, rats, monkeys, hamsters, frogs, rabbits and the like. I wish you. Preference is given to feeder cells from human or mouse species.

Another important criterion for obtaining hBS cell lines meeting general requirements is the conditions under which blastocysts are cultured. Thus, stem cell lines derived from the blastocysts can be propagated by incubating with feeder cells having a density of less than about 60,000 cells / cm 2, for example less than about 55,000 cells / cm 2 or less than about 50,000 cells / cm 2. In certain embodiments, proliferation of stem cell lines derived from blastocysts comprises culturing the stem cells with feeder cells having a density of about 45,000 cells / cm 2. This value applies to the use of mouse feeder cells and it is contemplated that suitable densities can also be found for other feeder cells. Based on our findings, one skilled in the art will be able to find such suitable densities. The feeder cells can be inactivated by mitosis to avoid their unnecessary growth.

Stem cell lines derived from blastocysts obtained by the above-described establishment method maintain self-renewal and pluripotency for a suitable period of time, and thus they are stable for a suitable period of time. The term "stable" in the text is intended to indicate the proliferative capacity of an undifferentiated state for at least 21 months when proliferated on embryonic feeder cells inactivated by mitosis.

Stem cell lines obtained by the above-described establishment method satisfy general requirements. Thus, the cell line

i) show proliferative capacity undifferentiated for at least 21 months when proliferated on embryonic feeder cells inactivated by mitosis;

ii) shows normal euploid chromosome karyotypes,

iii) retaining the potential to develop into derivatives of all types of germ layers both in vitro and in vivo,

iv) around keratin sulfate / chondroitin sulfate cells recognized by the following molecular markers OCT-4, alkaline phosphatase, carbohydrate epitopes SSEA-3, SSEA-4, TRA 1-60, TRA 1-81, and monoclonal antibody GCTM-2 At least two of the protein cores of the matrix protein glycans,

v) does not exhibit molecular marker SSEA-1 or other differentiation markers,

vi) teratomas form in vivo upon injection into immunocompromised mice, maintaining their pluripotency,

vii) can differentiate.

Undifferentiated hBS cells obtained by the method described above are defined by the following criteria; The cells were isolated from fertilized oocytes, ie blastocysts, before human implantation and exhibited their ability to proliferate undifferentiated upon proliferation on feed cells inactivated by mitosis; The cells exhibit normal chromosomal karyotypes; The cells were assigned to markers typical for undifferentiated hBS cells, such as OCT-4, alkaline phosphatase, carbohydrate epitope SSEA-3, SSEA-4, TRA 1-60, TRA 1-81, and monoclonal antibody GCTM-2. Expresses the protein core of the matrix protein glycan periphery of keratin sulfate / chondroitin sulfate cells, and does not show any expression for carbohydrate epitope SSEA-1 or other differentiation markers. Moreover, in vitro and in vivo (teratoma) pluripotency tests demonstrate differentiation into all germline derivatives.

In accordance with the above, the method shows i) proliferative capacity in the undifferentiated state for at least 21 months when proliferated on mitotically inactivated embryonic feeder cells, ii) shows normal euploid chromosomal karyotypes, and iii) ex vivo. And retain the potential to develop into derivatives of all types of germ layers both in vivo, and iv) the following molecular markers OCT-4, alkaline phosphatase, carbohydrate epitopes SSEA-3, SSEA-4, TRA 1-60, TRA 1-81, and at least two of the protein cores of the matrix proteinglycan periphery of keratin sulfate / chondroitin sulfate cells recognized by monoclonal antibody GCTM-2, v) without showing molecular marker SSEA-1 or other differentiation markers, vi) essentially pure agent of pluripotent human BS cells capable of differentiating and forming teratomas in vivo upon injection into immunocompromised mice, maintaining their pluripotency It was proved.

Procedures for detecting cellular markers can be found in Gage, F.H., Science, 287: 1433-1438 (2000). The procedure is well known to the skilled person and includes methods such as immunoassay or RT-PCR using antibodies directed against cell markers. In the following, methods for detecting cell markers, hybridization methods, karyotyping, methods for measuring telomerase activity and teratoma formation are disclosed. These methods can be used to investigate whether hBS cells obtained according to the above established methods meet the above mentioned criteria.

Immunohistochemistry

HBS stem cells maintained in culture are typically monitored for their differentiation status. Cell surface markers used to monitor the undifferentiated hBS cells are SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81. Human BS stem cells are fixed in 4% PFA and then made permeable with 0.5% Triton X-100. After washing and blocking with 10% milk powder, the cells are incubated with the primary antibody. After extensive washing, cells are incubated with secondary antibodies and nuclei are visualized by DAPI staining.

Alkaline Phosphatase

The activity of alkaline phosphatase is commercially available and is measured using the kit according to the manufacturer's instructions (Sigma Diagnostics).

Oct-4 RT- PCR

MRNA levels of the transcription factor Oct-4 are measured using RT-PCR and gene specific primer sets (5'-CGTGAAGCTGGAGAAGGAGAAGCTG, 5'-CAAGGGCCGCAGCTTACACATGTTC) and GAPDH (5'-ACCACAGTCCATGCCATCAC, 5'-TCCACCACCCTGTTGCTGTA) as housekeeping genes. .

Fluorescence equal Reaction system Hybridization (FISH)

At least one chromosome in one round of FISH is selected as a chromosome specific probe. This technique allows for the detection of numerical genetic deviations (if any). For this assay CTS uses commercially available kits containing probes for chromosomes 13, 18, 21 and sex chromosomes (X and Y) (Vysis, Inc, Downers Grove, IL, USA). More than 20 nuclei are analyzed for each cell line. The cells are resuspended in Carnoy fixative and dropped on positively charged glass slides. Probe LSI 13/21 is mixed with LSI hybridization buffer and added to the slide and covered with a cover slip. Probe CEP X / Y / 18 is mixed with CEP hybridization buffer and added to another slide in the same manner. The denaturation is performed at 70 ° C. for 5 minutes and then hybridized at 37 ° C. for 14-20 hours in a moisture chamber. Following a three step wash procedure, the nuclei are stained with DAPI II and the slides are analyzed under an inverted microscope (CytoVision, Applied Imaging) equipped with suitable filters and software.

Karyotyping

Karyotyping allows all chromosomes to be studied in a direct way, giving a lot of information, and detecting both numerical and larger structural deviations. To detect mosaics, more than 30 karyotypes are needed. However, this technique is very time consuming and technically complex. To improve the assay conditions, the mitotic index can be generated by colcemid, a synthetic homologue to colchicine and a microtubule-stabilizer that stops cells in the medium phase, but still requires a large cell supply (6 × 10 ⁶ cells / assay). The cells are incubated for 1 to 2 hours in the presence of 0.1 μg / ml colsemid, then washed with PBS and trypsinized. The cells are harvested for 10 minutes by centrifugation at 1500 rpm. The cells are fixed using ethanol and glacial acetic acid and the chromosomes are visualized using altered Wrights staining.

Genome by comparison Hybridization

Genome hybridization by comparison (CGH) is a complement to karyotyping. CGH provides higher analytical capacity for chromosomes and is less technically challenging. Isolated DNA is nicktranslate in a mixture of DNA, A4, Texas Red-dUTP / FITC 12-dUTP, and DNA polymerase I. Agarose gel electrophoresis is performed to control the size (600-2000 bp) of the DNA fragments produced. Test and control DNA are precipitated and resuspended in a hybridization mixture containing formamide, dextran sulfate and SSC. Hybridization is carried out on glass slides modified with heavy medium for 3 days in a moisture chamber at 37 ° C. After extensive washing, a drop of antifade fixation mixture (vectashield, 0.1 μg / ml DAPI II) is added and the slide is covered with a cover slip. Slides are then evaluated using a phase analysis system under a microscope.

Telomerase  activation

Since high activity has been defined as the reference for 6 hBS cells, telomerase activity is measured in these hBS cell lines. Telomerase activity is known to decrease one after another as cells reach a more differentiated state. Thus the quantitative analysis of the activity must be related to earlier passages and control samples, and the assay can be used as a detection tool for differentiation. This method, in which the telomerase PCR ELISA kit (Roche) uses the internal activity of telomerase, amplifies the product by polymerase chain reaction (PCR) and detects it by enzyme-linked immunosorbent assay (ELISA). The analysis is carried out according to the manufacturer's instructions. Results from this assay typically show high telomerase activity (> 1) against hBS cells.

The cell line retains its pluripotency and forms teratomas in vivo when injected into immunocompromised mice. In addition, in vitro the cells can form hBS cell derived bodies. In both models, cell characteristics for all germ layers can be found.

Teratoma Formation in Immunodeficiency Mice

One method of analyzing whether a human BS cell line maintains pluripotency is to xenograft the cells into immunodeficient mice to obtain teratomas that are tumors. The various types of tissues found in these tumors should all exhibit three germ layers. The reports showed various tissues in tumors from xenograft immunodeficient mice, for example rhabdomyocartilage and bone (mesoderm), intestine (endoderm), and neural rosette (ectoderm). In addition, much of the tumor consists of collapsed tissue.

Severe complex immunodeficiency (SCID) -mouse (a strain lacking B- and T-lymphocytes) is used for teratoma formation analysis. Human BS cells are surgically placed under the testicle or kidney membrane. In the testicles or kidneys, hBS cells are translated in the range of 10,000 to 100,000 cells. Ideally, 5 to 6 mice are used for each cell line at a time. Preliminary results show that female mice are more stable postoperatively than male mice and that xenografts to the kidneys are as effective as testes in tumor development. Therefore, female SCID-mouse teratoma model is preferred. Tumors are usually palpable after approximately 1 month. The mice are killed after 1-4 months and tumors are excised and fixed for paraffin- or freeze-incision. The tumor tissue is subsequently analyzed by immunohistochemistry. Use markers specific to all three germ layers. The markers currently used are: human E-cadherin, α-smooth muscle actin (mesoderm), α-fetoprotein (endoderm), and β-III-tubulin (ectoderm) for differentiation between mouse tissue and human tumor tissue ). In addition, hematoxylin-eosin staining is performed on the general form.

The establishment method is described in the following "establishment example". These examples are included in the present invention for purposes of illustration and are not intended to limit the scope of the present invention in any way. The general methods disclosed herein are well known to those skilled in the art and all reagents and buffers are readily commercially available or readily prepared according to well-established protocols by those skilled in the art. All incubations were performed under 37 ° C., CO ₂ atmosphere.

One suitable medium used is referred to as "BS-cell medium" or "BS-medium", which medium is 20% KNOCKOUT® serum supplement, and the following components at each final concentration: penicillin KNOCKOUT supplemented with 50 units / ml, streptomycin 50 μg / ml, non-essential amino acid 0.1 mM, L-glutamine 2 mM, β-mercaptoethanol 100 μM, human recombinant bFGF (basic fibroblast growth factor) 4 ng / ml Dulbecco's modified Eagle's medium.

Another suitable medium is “BS cell body medium” and may include the following: 20% KNOCKOUT® serum supplement, and the following components at each final concentration: 50 units / ml penicillin, strepto KNOCKOUT® Dulbecco's modified Eagle's medium supplemented with 50 μg / ml of mycin, 0.1 mM of essential amino acid, 2 mM of L-glutamine, and 100 μM of β-mercaptoethanol.

In the present text, the term "stable" is intended to indicate the proliferative capacity of an undifferentiated state for at least 21 months when proliferated on embryonic feeder cells inactivated by mitosis.

Test Example

Test Example  One

Spontaneously hatched From blastocyst  Establishment of essentially pure preparation of undifferentiated stem cells

Human blastocysts were obtained from frozen or fresh human in vitro fertilized embryos. Spontaneously hatched blastocysts were loaded with hBS cell medium [20% KNOCKOUT® serum supplement, and 0.125 mg / ml of hyaluronic acid, the following components at each final concentration: penicillin 50 units / ml, streptomycin 50 Modified KNOCKOUT Dulbecco supplemented with μg / ml, non-essential amino acid 0.1 mM, L-glutamine 2 mM, β-mercaptoethanol 100 μM, human recombinant bFGF (basic fibroblast growth factor) 4 ng / ml Eagle medium] directly on feeder cells (EF). After blastocysts were plated on the EF cells, growth was monitored when the colonies were large enough via manual passage culture approximately 1 to 2 weeks after the smears. Genus cell mass cells were excised from other cell types and expanded by proliferation on new EF cells.

Test Example  2

With full transparent band From blastocyst  Establishment of essentially pure preparation of undifferentiated stem cells

For blastocysts with complete clear zones, the zones were cut using simple pronase (10 U / ml, Sigma) culture in rS2 (ICM-2) medium (Vitrolife, Gothenburg, Sweden), after which the blastocysts were cut. Placed directly on EF cell layer in hBS medium supplemented with hyaluronic acid (0.125 mg / ml).

Test Example  3

Alkaline Phosphatase  For histochemical staining

Cells were harvested for RT-PCR and tissue (alkaline phosphatase) and immunocytochemical analysis (see below). RNA isolation and RT-PCR. Whole cell RNA was prepared according to the manufacturer's recommendations using the Rneasy Mini Kit (Qiagen). cDNA analysis was performed using AMV First Strand cDNA Synthesis Kit (Roche) for RT-PCR and PCR using Platinum Taq DNA Polymerase (Invitrogen). Histochemical staining for alkaline phosphatase was performed according to manufacturer's recommendations using a commercially available kit (Sigma).

Test Example  4

Preparation and Culture of hBS Cell Line

Mouse embryo fibroblast feeder cells were placed on an EMFI-medium, ie 10% FCS (calf fetal serum), 0.1 μM β-mercaptoethanol, 50 units / ml penicillin, 50 μg / ml streptomycin, and 2 mM L on tissue culture dishes. Cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with Glutamine (GibcoBRL). Feeding cells were mitotically inactivated with mitomycin C (10 μg / ml, 3 hours). Human BS cell-colonies were expanded onto mouse embryonic fibroblast feeder cells inactivated by manual excision.

Human BS cells were prepared in hBS-cell medium, ie 20% KNOCKOUT® serum supplement, and the following components at each final concentration: 50 units / ml penicillin, 50 μg / ml streptomycin, 0.1 mM essential amino acid, Mitosis in tissue culture dishes with modified Eagle's medium from KNOCKOUT® Dulbecco supplemented with 2 mM L-glutamine, 100 μM of β-mercaptoethanol, 4 ng / ml of human recombinant bFGF (basic fibroblast growth factor) Cultured on inactivated mouse embryo fibroblast feeder cells. On day 7 after passaging the colonies are large enough to produce BS cell bodies.

BS cell colonies were cut into 0.4 × 0.4 mm pieces with glass capillaries and the BS cell sieve medium, ie 20% KNOCKOUT® serum supplement, and the following components at each final concentration: 50 units / ml penicillin, strepto On a non-adherent bacterial culture dish containing modified Eagle's medium from KNOCKOUT® Dulbecco supplemented with 50 μg / ml of mycin, 0.1 mM of essential amino acid, 2 mM of L-glutamine and 100 μM of β-mercaptoethanol Put on. The BS cell bodies, including hBS cells with cysts, formed over a period of 7-9 days.

Test Example  5

Passage culture of hBS cells

Before passaging, hBS cells are photographed using a Nikon Eclipse TE2000-U inverted microscope (10 × objective) and a DXM 1200 digital camera. Colonies are passaged every 4 to 5 days. The colonies are of sufficient size to passaging when they can be cut into pieces (0.1 to 0.3 x 0.1 to 0.3 mm). The first time the cells are passaged the cells proliferate for 1-2 weeks and can be cut into approximately 4 pieces.

The colonies are focused one by one under a stereo microscope and cut into a checkerboard pattern according to the size. Only passages with a uniform inner structure are carried out. Colonies from each compartment are collected with a knife, sucked into capillaries and placed on new feeder cells (up to 4 days of age). Place 10-16 cells evenly on all new IVF-plates. The dish is left for 5-10 minutes to allow the cells to adhere to the feeder cells and then placed in the incubator. The hBS medium is changed three times a week. If colonies are passaged, the medium is changed in that particular week. Typically, “half exchange” is performed, meaning that only half of the medium is sucked in and replaced with an equal amount of fresh conditioned medium. In some cases, the entire medium may be replaced.

Test Example  6

hBS cells Supermagnetization

Colonies with suitable undifferentiated form from this cell line are cut for passaging. 100-200 ml of liquid nitrogen is sterile filtered through a sufficient amount of cold tube. Prepare two solutions A and B (A: 800 μl cold PBS with 1M trehalose, 100 μl ethylene glycol and 100 μl DMSO, B: 600 μl cold PBS with 1M trehalose, 200 μl ethylene glycol and 200 μl DMSO) colonies are left in A for 1 minute and left in B for 25 seconds. Closed straw is used to store the frozen colonies. After transferring the colonies to the straw, they are placed directly in a cold tube with sterile filtered nitrogen.

Test Example  7

Embryonic Mouse Nutrition EMFi A) cellular Seeding

Cells are inactivated with EMFi medium containing mitomycin C by incubation at 37 ° C. for 3 hours. IVF-plates are coated with gelatin. The medium is aspirated and the cells washed with PBS. The cells are detached by replacing PBS with trypsin. After incubation, trypsin activity is stopped with EMFi medium. The cells are then harvested by centrifugation, diluted 1: 5 with EMFi medium, and counted in a Burker chamber. The cells are diluted to a final concentration of 170K cells / ml EMFi medium. The gelatin in the IVF-plate is replaced with 1 ml of cell suspension and placed in the incubator. The EMFi badge is changed on the same day after seeding.

hBS cell feeder cells Supported  Efficient Migration from Feeding Systems to a Feeder-Free Culture System, and Long-term Proliferation of hBS Cells in the Absence of Feeding Cells

The hBS cells used in the present invention can be cultured in a feeder cell free culture system, which method is advantageous over known methods in which migrated cells are stable for up to at least 10 passages. Richard et al. Studies showed that the hBS cell line could not be propagated undifferentiated for at least 6 passages on a cell-free substrate, for example Matrigel ^™ . However, hBS cells were stable for up to 35 passages on Matrigel ^™ , indicating that the expression of markers for undifferentiated hBS cells still remained roughly comparable even after freeze / thaw and growth rate cycles. Moreover, significantly more viable colonies were observed on day 2 after smearing, with mechanical dissociation comparable to dissociation by enzymes. An important step seems to be the initial migration of hBS cells into the feeder cell free culture system. Thus, disclosed below is a method of delivery of hBS cells to a feeder cell free culture system, wherein the hBS cells are mechanically cleaved from the feeder cells. In the absence of feeder cells of the present invention, only the central portion of each colony is used, whereas in previous studies such as Xu, the whole colony was desorbed by enzyme treatment, wherein the culture was contaminated by feeder cells. There is a risk. Moreover, the use of enzymes at the very delicate stages of moving feeder cell cultured hBS cells to the feeder cell free surface can inactivate important surface molecules involved in cell adhesion and growth. Main components in Matrigel ^™ are extracellular matrix proteins such as collagen type IV and laminin. Activation of cell surface integrins upon binding to extracellular matrix proteins is believed to be an important step in the regulation of cell adhesion, survival and proliferation. For example, integrin alpha 1 plays a unique role in regulating cell proliferation both in vivo and ex vivo in collagen substrates, among collagen receptors. Putatively, laminin-specific receptors formed by integrins α6 and β1 that are highly expressed by hBS cells may also play an important role in adhesion of hBS cells to the substrate surface. Thus, one possibility is that some of the surface receptors important for adhesion or survival may be negatively affected by rough early collagenase IV treatment before cells adapt to the new surface.

In the examples of the present invention different transfer techniques of hBS cells to the feeder cell free environment were investigated by mechanical or enzymatic dissociation with respect to cell attachment, viability and proliferation. Furthermore, the method was developed to promote long-term proliferation and large scale production of a homogeneous population of undifferentiated hBS cells. The use of conventional cryopreservation techniques for freeze / thaw of hBS cells was also investigated.

Migration of hBS cells to trophoblast-free proliferation

Following ablation of the genus cell mass, the genus cell mass cells are incubated with feeder cells to obtain a blastocyst-derived stem (BS) cell line. After obtaining the hBS cell line, the cell line is optionally propagated to expand the amount of the cell.

Prior to propagating the hBS cells in the feeder cell free system, the hBS cells can be transferred to the feeder cell free system.

As previously mentioned in the present invention and demonstrated in the feeder cell free example, an important success factor in the proliferation of hBS cells is the method of transferring hBS cells from feeder cell culture systems to feeder cell free culture systems. Therefore, the hBS cells should be transferred to a feeder cell free culture system by mechanical excision (which can be performed using free capillaries as cutting tools). As shown in the examples of the present invention, mechanical ablation attaches cells to Matrigel ^™ much more efficiently, which is more rapid proliferation compared to enzyme treated cultures and the cells were much more stable during passaging. Thus, the method of migrating HS cells according to the present invention does not require enzyme treatment. As shown in the examples of the present invention, cells cultured and propagated under vegetative cell conditions have a mitotic index similar to cells proliferated under vegetative cell conditions.

The proliferation of stem cell lines derived from blastocysts has many advantages in culturing the hBS cells without feeder cells, for example, without the need for continued production of feeder cells, and extending the production of hBS cells to a commercial scale of production. May be easier, and there is no risk of DNA transfer or other infection from feeder cells, thereby culturing the stem cells under feeder cell-free growth conditions.

Thus, the migration and proliferation step under vegetative cell absence conditions may comprise the following steps:

a) transferring stem cells derived from blastocysts from feeder cells to feeder cell free cultures by mechanical treatment,

b) optionally, culturing stem cells derived from said blastocysts in suitable growth medium and / or on suitable support substrates under vegetative cell free growth conditions, and

c) optionally, passage of stem cell lines derived from blastocysts every 3 to 10 days by enzyme and / or mechanical treatment.

Typically, all steps i) to iii) are included.

Transfer of hBS cells from feeder cell culture system to feeder cell free culture system

The migration step was found to be an important step as mentioned above. Therefore, the transfer must be performed by mechanical dissociation or mechanical ablation of the cells in the nutrient culture system. Such mechanical treatment may be performed by any suitable cutting tool, for example a tool having a sharp end and a size suitable for cutting. The tool can be made of any suitable material, for example plastic or glass, examples of suitable tools having an angle of 25 degrees and 200 or 300 micrometer lumens, cutting, manipulating and cutting hBS colonies or portions of hBS colonies and A cutting tool that is a sharp sterile glass capillary designed for transfer. It is produced by Sweden Lab International AB (Billdal, Sweden).

The hBS cells to be transferred are colonies of hBS cells and the fragments are cut from the center of the colonies and suspended in suitable media as cell populations. The cell population may be mechanically subjected to one or more times, eg, the cell population is at least 50% of the original colony size, for example at most about 40%, at most about 30%, at most about 20%, at most about 10% or at about 5 Dissociate until it has a size of% or less. The size is measured, for example, as the diameter of the population or colony, respectively.

In the absence of feeder cells of the present invention, conditions suitable for the migration process are provided. These conditions can of course be varied within suitable limits that are within the knowledge of those skilled in the art.

Nutrient cell absence Example  One

Conditions for feeder cell free cultures Vitrohes ^TM Medium (k- Vitrohes ^TM -Medium)

For the preparation of mEF cells for conditioning of VitroHES ^™ -medium, fusion monolayers of mEF cells (two passages) were treated with mitomycin C and gelatin (0.1%; Sigma) coated culture flasks in 1% penicillin / strepto Mycin (PEST; 10000 U / mL), 10% Fetal Bovine Serum (FBS) and 2 mM GLUTAMAX ^™ -I Supplement (200 mM) (these reagents are all available from GibcoBRL / Invitrogen, Carlsbad, CA, USA) ) Was seeded at a concentration of 59,000 cells / cm 2 in Dulbecco's modified Eagle's medium (D-MEM). After 24 hours of incubation and washing once with PBS (GibcoBRL / Invitrogen), the medium was discarded and replaced with VitroHES ^™ -medium (0.28 mL / cm 2) for a 24 hour conditioning period. The conditions VitroHES ^™ medium (k-VitroHES ^™ medium) were collected from the same mEF culture (two passages) up to three times daily and sterile filtered using a 0.2 μm low protein binding filter (Sarstedt, Landskrona, Sweden). It was. The k-VitroHES ^™ -medium was fresh or used after freezing at −20 ° C. and supplemented with 4 ng / ml bFGF (GibcoRL / Invitrogen) before use. The k-VitroHES ^™ medium can be used for up to one week when stored at + 4 ° C. When stored for two months at -20 ° C, no signs of reduced bioreactivity were detected during use.

Nutrient cell absence Example  2

Migration to Adoptive Growth Conditions in HBS Cell Lines

Initial hBS cell lines were maintained on mitomycin C treated mouse feeder cells in 10-50 passages cultured and cultured in VitroHES ^™ -medium supplemented with 4 ng / ml human basal fibroblast growth factor (bFGF).

Two different techniques were evaluated for migration from feeder cell cultures of hBS cells to Matrigel ^™ coated plates, one of which is mechanical dissociation and the other is collagenase treatment. The hBS cells were cut into square pieces, the center specimens of the colonies, using a stem cell cutting tool (Swemed Lab AB, Billdal, Sweden) and carefully detached to transfer the cells to HBSS solution. The stem cell tool is a sharp sterile glass capillary with an angle of 25 degrees and between 200 and 300 micrometer lumens and designed for cutting, manipulating and moving hBS colonies or portions of hBS colonies. It is produced by Sweden Lab International AB (Billdal, Sweden).

To collagenase  Enzyme treatment (for control)

After washing in HBSS, the cell populations were transferred to collagenase IV solution (200 U / ml; Sigma) for enzyme dissociation. The cells were incubated for 30 minutes in 37 ° C. and 5% CO ₂ . During the incubation period, repeated mechanical dissociation was performed using a pipette and the dissociation process was monitored by an inverted microscope. After this incubation period, the cell suspension was pelleted (400 G for 5 minutes) and washed once in KnockOut ^™ D-MEM (GibcoBRL / Invitrogen) before resuspending in k-VitroHES medium.

Mechanical dissociation according to the invention

After washing in HBSS, the cell culture was mechanically carefully dissociated using a 1-ml auto pipette. The dissociation process was carried out in which the size of the cell population was caused by collagenase IV treatment (the colonies were transferred to collagenase IV solution (200 U / ml) and washed with HBSS to initiate enzyme dissociation after washing in HBSS as described above). Completion when representing about 1/10 to 1/20 (average 20,000 cells / original colony) of the original colonies corresponding to. For these two different techniques, cells were seeded in four wells each and incubated in 37 ° C., 5% CO ₂ . Each experiment was repeated four times, with the same amount of cells seeded each time. After 2 and 6 days, colony size and number were calculated.

Nutrient cell absence Example  Result of 1 and 2

In order to optimize the migration of feeder cells from feeder cells in hBS culture to feeder cell free conditions, two different techniques were evaluated, where one technique is mechanical dissociation and the other is enzyme dissociation. Mechanical dissociation resulted in more efficient cell attachment and faster proliferation to Matrigel ^™ compared to enzyme treated cultures. When comparing mechanical dissociation and enzyme dissociation (FIG. 5), a significantly larger number of viable colonies were observed on day 2 after smearing. The total immunity of all colonies generated on Matrigel ^™ after dissociation by each of the two different techniques was compared (P <0.001). Furthermore, on day 6 after smearing the total colony area in the mechanically dissociated cultures was significantly increased compared to the enzyme dissociated cultures (P = 0.036).

Nutrient cell absence Example  3

Matrigel ^TM  And passage culture of hBS cells cultured on

Four different cell lines SA 002, AS 038, SA 121 and SA 167 were used for all experiments. Cell lines were grown on Matrigel ^™ up to 35 passages and the morphological appearance and other hBS characteristics remained unchanged even after the freeze / thaw cycles. All cultures consisted of well defined colonies of hBS cells with no difference in morphological signs. After about 3-6 days, cells were taken up and passaged in medium and 1 ml of collagenase IV (200 U / ml) solution was added to each well and incubated for 15-20 minutes. In order to facilitate cell detachment from the surface, mechanical dissociation followed by incubation for an additional 15 minutes. Cells were then washed, resuspended in k-Vitro ^™ HES medium and seeded at a split ratio of 1: 2 to 1: 6 on Matrigel ^™ . The hBS culture was passaged every 5-6 days and the medium was exchanged every other day.

Nutrient cell absence Example  3 results

It was observed that enzymatic treatment with collagenase IV was required to detach colonies from the surface during passaging of hBS cells established on Matrigel ^™ . Enzyme treatment in subcultures was also found to provide increased proliferation rate after seeding compared to mechanical dissociation.

Nutrient cell absence Example  4

Matrigel ^TM  Cryopreservation and thawing of hBS cells cultured on

Four different cell lines SA 002, AS 038, SA 121 and SA 167 were treated with collagenase IV for 20-30 minutes prior to freezing to separate the cells from each other. After centrifugation, cells are contained in freezing medium (k-VitroHES ^™ -medium containing 10% DMSO, 30% serum supplement and 4 ng / ml of bFgF at a concentration of 1 x 10 ⁶ cells per ml of freezing medium) Moved to. The final cell suspension was a mixture of both single cells and cell populations. Cold tubes (0.5-1.0 ml of cell suspension) were quickly transferred to Nalgene freezing vessels for storage overnight at -80 ° C or for at least 2 hours before long term storage in liquid nitrogen.

Thawing of hBS Cells

The k-VitroHES ^™ -medium must be prepared and preheated before thawing the cells by placing the cold tube in a 37 ° C. water bath until all of the cell suspension has thawed. The cell suspension was transferred to the preheated medium for 5 minutes before centrifugation (400 G, 5 minutes). Matrigel ^™ thin coated (BD) wells were rehydrated by adding 1 ml of k-VitroHES ^™ -medium to the wells and incubated at 37 ° C. for 30 minutes. Cell pellets were resuspended in k-VitroHES ^™ -medium and transferred to 24- or 6-well Matrigel ^™ plates.

Nutrient cell absence Example  5

Characterization of hBS cells cultured in the absence of feeder cells

All characterization experiments were performed after the establishment and freeze / thaw cycles on Matrigel ^™ .

Immunocytochemistry:

Cultures were passaged as described above, seeded in 6- or 24-well Matrigel ^™ plates and incubated for 6 days before immunostaining. Cultures were washed with PBS, fixed with 4% formaldehyde (HistoLab, Gothenburg, Sweden) for 15 minutes at room temperature and then washed three times with PBS again. Monoclonal primary antibodies used were SSEA-1, -3 and -4 (1: 200; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), Tra-1-60, Tra-1-81 (1: 200; Santa Cruz Biotechnology, Santa Cruz, CA), and polyclonal rabbit anti-phospho-histone H3 (1: 150; KeLab, Upstate). The primary antibody was incubated overnight at 4 ° C. and then visualized using a suitable Cy3- or FITC-conjugated secondary antibody (1: 300; Jackson ImmunoResearch Laboratories, West Grove, PA). The cultures were also incubated with 4'-6'-diimidino-2-phenylindole (DAPI; Sigma-Aldrich Sweden AB, Stockholm, Sweden) at a final concentration of 0.5 ug / ml for 5 minutes at room temperature for all cells. Nuclei were visualized. Stained cultures were rinsed and sampled using DAKO fluorescence sampling medium (Dakopatts AB, Alvsjo, Sweden) and visualized with an inverted fluorescence microscope (Nikon Eclipse TE2000-U). Alkaline phosphatase (AP) staining of Matrigel ^™ cultured hBS cells was performed according to the manufacturer's instructions using a commercially available kit (Sigma-Aldrich).

Telomerase  activation:

Matrigel ^™ cultured hBS cells were harvested, lysed and telomerase activity was analyzed by PCR-based ELISA (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions.

Karyotyping and FISH:

Matrigel ^™ propagated hBS cells designated for karyotyping were incubated for 1 to 3 hours in colsemid (0.1 μg / ml, Invitrogen, Carlsbad, Calif., USA), dissociated, fixed and placed on glass slides, Chromosomes were visualized using altered litz staining (# WS-32, Sigma). Preparation of the medium plate was carried out as previously described. For fluorescence in situ hybridization (FISH) analysis, a commercially available kit containing a probe for chromosomes 13, 18, 21 and sex chromosomes (X and Y) (MultiVysion ^™ PB Multicolour Probe Panel; Vysis, Inc. , Downers Grove, IL) was used according to the manufacturer's instructions. Slides were analyzed using an inverted microscope equipped with suitable filters and software (CytoVision, Applied Imaging, Santa Clara, Calif.).

Teratoma:

For teratoma formation experiments, immunodeficient SCID mice (CB-17 / IcrCrl-scidBR, Charles River Laboratories, Germany) were used. Matrigel ^™ propagated hBS colonies are desorbed from the surface by enzymes using collagenase IV (200 U / ml), mechanically dissociated into small cell aggregates, and approximately 50,000 to 100,000 cells / organs are injected under the kidney membrane It was. Control animals were treated with cold-PBS injection or litter primary brain cells. The animals were killed 8 weeks after injection and tumors were immediately fixed in 4% paraformaldehyde solution and paraffin embedded. For histological analysis, teratomas were made into small pieces of 8 μm and stained with Alcian Blue / Van Giesson.

RT- for Oct-4 expression PCR  analysis:

Total RNA was isolated from all four Matrigel ^™ cultured hBS cell lines using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized from 1 μg total RNA using AMV First Strand cDNA Synthesis Kit (Roche) and PCR was performed using platinum Taq DNA polymerase (Invitrogen). The PCR reaction included four initial stage-down cycles, two of which were repeated for all annealing temperatures, denaturing for 15 seconds at 94 ° C., annealing for 15 seconds at 66-60 ° C. and 72 ° C. Is an extension for 30 seconds. The following cycles included 35 repetitions with annealing temperature at 58 ° C. The forward and reverse primer sequences for Oct-4 have been described above. β-actin sequence was used as internal control (sense, 5'-TGGCACCACACCTTCTACAATGAGC-3 '; antisense, 5'-GCACAGCTTCTCCTTAATGTC-ACGC-3'; 400 bp product). The PCR product was sized by gel electrophoresis using 1.5% agarose gel. Human liver was used as a positive control for the PCR reaction and water as a negative control.

Nutrient cell absence Example  Results for 4 and 5

Cell lines SA 002, AS 038, SA 121 and SA 167 were frozen and thawed using cryopreservation techniques to see if any property changes could be found. After thawing all four cell lines survived and began to grow in a similar pattern on Matrigel ^™ coated plates.

The pluripotency and persistence of these four different hBS cell lines in the absence of feeder cells were demonstrated and compared with previous results for feeder cell cultures of each cell line. This characterization was performed by examining morphology, expression of undifferentiated markers, telomerase activity, karyotype and in vivo differentiation.

Immunocytochemistry:

SSEA-1 expression is all nutritional, as opposed to staining with antibodies to SSEA-3, SSEA-4, TRA-1-60 and TRA 1-80, which show the apparent positive immune response expected for pluripotent hBS cells. It was negative in hBS cell lines cultured in the absence of cells. Moreover, the cells showed high levels of AP reactivity in all four Matrigel ^™ propagated cell lines.

Telomerase  activation:

Assays were performed on three Matrigel ^™ cultured hBS cell lines (AS 038, SA 121 and SA 167). HBS cells cultured on the Matrigel ^™ were found to have high levels of telomerase activity.

Karyotyping and FISH:

Karyotyping was performed on two Matrigel ^™ cultured cell lines AS 038 and SA 121. Three of three cells from cell line AS 038 and 10 of 12 cells from cell line SA 121 were found to have normal human 46, XY karyotypes (FIG. 10). The remaining two cells from the SA 121 cell line expressed abnormal karyotypes 45, XY and 42, XY. However, karyotyping appears to be an event that normally occurs after prolonged culture for both feeder cell- and feeder-free hBS cell cultures. In this study, karyotyping on feeder cell cultured hBS cells was comparable to the results after Matrigel ^™ proliferation, suggesting that hBS cell karyotype remains normal and stable under the feeder cell absence conditions. FISH analysis was performed on two Matrigel ^™ propagated cell lines (SA 121 (XY) and SA 167 (XX)). Analysis was performed on chromosomes X, Y, 18, 13 and 21. More than 93% were normal for both cell lines tested. The results from the FISH assay were comparable to those from feeder cell cultured hBS cell lines.

Teratoma formation:

Teratoma formation was performed on two Matrigel ^™ cultured hBS cell lines, SA 167 and SA 002, with the result that the teratoma formed consisted of typical differentiated cells and tissues of all three germ layers (endoderm, mesoderm and ectoderm). This provides evidence that Matrigel ^™ propagated hBS culture maintains its pluripotency.

Oct-4 Expression: Oct-4 expression was high in all four cell lines cultured on Matrigel ^™ .

Nutrient cell absence Example  6

For hBS cells cultured on embryonic mouse feeder cells, Matrigel ^TM  Comparison of the mitotic index of hBS cells cultured in the absence of feeder cells on coated plates

Cell line SA 121 was incubated simultaneously for 3 days under feed cell free conditions on Matrigel ^™ coated plates and on embryonic mouse feeder cells. The number of mitotic cells was then quantified by nuclear immunoreactivity against phosphorylated histone H3. Mitosis indices were calculated in both cultures to compare the growth rate between feeder cell free and feeder cell cultured hBS cells.

Example  6 results

Mitotic indices were similar in cultures grown in feeder cell layer conditions and in the absence of feeder cells (Matrigel ^™ ). The doubling time of the feeder cell free culture was roughly the same as for feeder cell propagated hES cells (approximately 35 hours).

Claims (35)

i) transfer the cells to the first solution (solution A), ii) optionally culturing cells in said first solution, iii) transfer the cells obtained in step i) or ii) to a second solution (solution B), iv) optionally culturing cells in said second solution, v) transfer the cells obtained in step iii) or iv) to one or more closed straws having dimensions such that they can contain a volume of at least 20 μl, vi) sealing said at least one closed straw, vii) vitrify the one or more closed straws Including, the method of cell vitrification. The method of claim 1, The closed straw has dimensions of about 20 μl to about 250 μl, such as about 20 μl to about 225 μl, about 25 μl to about 200 μl, about 25 μl to about 175 μl, about 25 μl to about 150 μl, about 30 μl to about 125 μl, about 30 μl to about 100 μl, about 35 μl to about 75 μl, about 40 μl to about 50 μl. The method according to claim 1 or 2, How cells are BS cells or BS cell masters. The method according to any one of claims 1 to 3, How the cells master hBS cells or hBS cells. The method according to any one of claims 1 to 4, At least one of the first and second solutions comprises at least one cryoprotectant. The method of claim 5, wherein At least one cryoprotectant is selected from the group consisting of glycerol, trehalose, sucrose, ethylene glycol, DMSO, propanediol and / or mixtures thereof. The method according to claim 5 or 6, Wherein the first and second solutions contain one or more cryoprotectants which are the same or different. The method according to any one of claims 5 to 7, Wherein the concentration of one or more cryoprotectants in the first and second solutions is the same or different. The method according to any one of claims 5 to 8, Wherein the total concentration of cryoprotectant in the second solution (calculated as% v / v,% w / w or M) is greater than said concentration in the first solution. The method according to any one of claims 5 to 9, The cryoprotectant is trehalose. The method of claim 10, The concentration of trehalose is about 0.02 M to about 1 M, for example about 0.05 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.2 M to about 0.7 M, about 0.3 M to about 0.65 M, about 0.4 M to about 0.6 M, about 0.45 M to about 0.55 M. The method according to any one of claims 5 to 9, The cryoprotectant is sucrose. The method of claim 12, The concentration of sucrose is about 0.02 M to about 1 M, for example about 0.05 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.2 M to about 0.7 M, about 0.3 M to about 0.65 M, about 0.4 M to about 0.6 M, about 0.45 M to about 0.55 M. The method according to any one of claims 1 to 13, At least one of the first and second solutions comprises a viscosity modifier. The method of claim 14, The viscosity modifier is selected from the group consisting of Ficoll, Percoll, hyaluronic acid, albumin, polyvinyl pyrrolidone, alginic acid, gelatin and glycerol. The method according to claim 14 or 15, The viscosity modifier is Piccol. The method of claim 16, The picol has a concentration of about 150 mg / ml or less, for example about 100 mg / ml or less, about 50 mg / ml or less, about 25 mg / ml or less, about 15 mg / ml or less or about 10 mg / ml or less. The method according to any one of claims 14 to 17, Wherein the first and second solutions contain one or more viscosity modifiers, identical or different. The method according to any one of claims 14 to 18, Wherein the concentration of one or more viscosity modifiers in the first and second solutions is the same or different. The method according to any one of claims 1 to 19, At least one of the first and second solutions is an aqueous solution. The method according to any one of claims 1 to 20, A method comprising step ii). The method of claim 21, The incubation is carried out for a period of 5 seconds to about 20 minutes, for example about 10 seconds to about 15 minutes, about 15 seconds to about 10 minutes, about 20 seconds to about 7.5 minutes, about 30 seconds to about 5 minutes, about 40 seconds. To about 4 minutes, about 50 seconds to about 3 minutes, about 30 seconds to about 2 minutes, about 45 seconds to about 1.5 minutes, or about 1 minute. The method according to any one of claims 1 to 22, A method comprising step iv). The method of claim 23, The culture is carried out for a period of about 5 seconds to about 10 minutes, for example about 10 seconds to about 7.5 minutes, about 10 seconds to about 5 minutes, about 15 seconds to about 4 minutes, about 15 seconds to about 3 minutes, about 15 And at about 37 ° C. for a period of seconds to about 2 minutes, about 20 seconds to about 1 minute, about 5 seconds to about 1 minute, about 5 seconds to about 30 seconds, or about 10 seconds to about 30 seconds. The method of claim 23 or 24, The incubation is carried out at about 37 ° C. for up to about 30 seconds. The method according to any one of claims 1 to 25, At least about 50%, for example at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, Or at least about 95% is viable after devitrification and incubation in a suitable medium. 27. A cell that has undergone vitrification by the method as defined in any one of claims 1 to 26. The method according to any one of claims 1 to 26, viii) subjecting the at least one vitrified closed straw to an environment having a temperature from about room temperature to about 40 ° C. during the time that the contents of the closed straw are thawed, ix) opening said at least one closed straw, x) subjecting the cells contained in the one or more open straws to a washing process using a third solution (solution C), xi) optionally transfer the washed cells obtained from step x) to a fourth solution (solution D), xii) optionally culturing cells in said fourth solution, xiii) optionally seeding the cells from xii) from the fourth solution and seeding the cells onto feeder cells, xiv) optionally further culturing the cells Also comprising de-supermagnetization by a method comprising a. The method of claim 28, A process comprising steps xi), xiii) and xiv). The method of claim 29, And also step xii). The method according to any one of claims 28 to 30, The third and / or fourth (if appropriate) solution comprises one or more cryoprotectants. The method of claim 31, wherein At least one cryoprotectant is selected from the group consisting of glycerol, trehalose, sucrose, ethylene glycol, DMSO, propanediol and / or mixtures thereof. The method of claim 32, At least one cryoprotectant is glycerol, trehalose, sucrose or mixtures thereof. The method of claim 33, wherein The cryoprotectant concentration is about 0.02 M to about 1 M, for example about 0.05 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.15 M to about 0.5 M, about 0.2 M to about 0.4 M. The method according to any one of claims 28 to 34, wherein Wherein the concentration of cryoprotectant in the third solution is greater than the concentration of cryoprotectant in the fourth solution, if appropriate.

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