ES2319714B1 - CELLULAR CRIOPRESERVATION BY VITRIFICATION WITH LOW CRIOPROTECTOR CONCENTRATIONS. - Google Patents

Abstract

Cell cryopreservation by vitrification with low concentrations of cryoprotectant.

The object of the present invention relates to a new cell cryopreservation procedure, which achieves the vitrification of biological samples with the presence of losses Cryoprotectant concentrations. The procedure is based on the use of forced convection in order to increase the heat transfer and achieve very high cooling rates and  overheating that prevents nucleation and growth of ice crystals

Description

Cell cryopreservation by vitrification with low concentrations of cryoprotectant.

Object of the invention

The object of the present invention relates to a new cell cryopreservation procedure, which achieves the vitrification of biological samples with the presence of losses Cryoprotectant concentrations. The procedure is based on the use of forced convection in order to increase the heat transfer and achieve very high cooling rates and  overheating that prevents nucleation and growth of ice crystals

State of the art

Two techniques are currently developed, with a multitude of variants, with the aim of achieve cell preservation: slow cooling and vitrification

Slow cooling is, by far, the most used procedure It is based on the rate control of cooling in order to create a delicate balance between the different factors that cause cell damage, among which they find icing, fractures and excessive dehydration of the cell.

In recent years another technique has appeared, vitrification, which greatly improves the technique of slow cooling, because it completely eliminates the formation of ice crystals, which represent the greatest obstacle to the cryopreservation of living cells. Currently, vitrification is considered as the future of cellular cryopreservation [ Gábor Vajta, Masashige Kuwayama, Improving cryopreservation systems, Theriogenology 65 (2006), 236-244 ]. However, the technique still needs to be improved for many cell types, such as oocytes, in which its vitrification and subsequent fertilization has so far resulted in only 16 births of children [ David K. Gardner, In vitro fertilization, a practical approach, Taylor &Francis; Edition: 1 (September 29, 2006 ). Vitrification of stem cells, another of the most interesting cell types of cryopreservation, of great potential for regenerative medicine, has just begun to be put into practice [ SS Gouk, CKF Tan, MP Hande, A. Poonepalli, GS Dawe and Lilia L. Kuleshova, Protein and serum free vitrification of neural item cells, Criobiology, Volume 53, Follow 3, December 2006, Page 389 ] and great experimentation will be necessary before it can be used efficiently.

Vitrification is a process by which a liquid solidifies in a non-crystalline phase (vitreous phase) with a rapid decrease in temperature and an increase in viscosity. Several factors affect the probability of getting a  adequate vitrification: sample viscosity, rhythm of cooling and heating, and sample volume [S. Latchkey, A. Arav, Measurement of essential physical properties of vitrification solutions, Theriogenology 2007; 67; 81-89].

In order to increase the viscosity of the sample, the so-called vitrifying agents, also called cryoprotectants, are used because of their relationship with cellular protection. With some of them, such as dimethylsulfoxide or ethylene glycol, good results have been achieved [ (Rall WF, Fahy GM: Ice-fre cryopreservation of mouse. Embryos at -196 by vitrification. Nature 1985; 313: 573-. 575); (T. Takahashi, A. Hirsh, EF Erbe, JB Bross, RL Steere and RJ Williams Vitrification of human monocytes, Cryobiology, Volume 23, Issue 2, April 1986, Pages 103-115 )]. However, the high concentrations necessary for these cryoprotective agents to achieve vitrification result in high toxicity that prevents their viability in many cell cryopreservation protocols.

The increase in the rate of cooling and heating increases the probability of adequate vitrification, and makes lower concentrations of cryoprotective agents necessary. The most used technique to achieve high cooling rates has been the immersion of the sample in liquid nitrogen. It is necessary to achieve efficient heat transmission between liquid nitrogen and cellular material, to maximize the cooling rate. With this objective, different containers have been applied to introduce cells in liquid nitrogen, among which we find copper grids used in electron microscopy [ Martino A, Songsasen N, Leibo S. P, Developing into blastocysts of bovine oocytes cryuopreserved by ultrarapid cooling, Biol Reprod 1996; 54; 1059-1069 ,], OPS capillaries [ Vajta, G, Booth PJ, Holm P, Greve T, Callesen H, Succesful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method , Cryo-Letters 1997; 18; 191-195 ] (Open Pulled Straws, conventional PVC capillaries stretched to have an inside diameter of 0.800 mm and a wall thickness of 0.075 mm) or the so-called cryoloops [ Lane M, Schoolcraft WB , Gardner DK, Vitrification of mouse and human blastocysts using a novel ctyoloop container-less technique, Fertil Steril, 1999; 72; 1073-1078 ], nylon ties in which a solution film containing the cells is suspended. However, the cooling rate achieved by these procedures still requires a high concentration of cryoprotectants to produce vitrification [ Gábor Vajta, Masashige Kuwayama, Improving cryopreservation systems, Theriogenology 65 (2006), 236-244 ]. To achieve better results in vitrification you can use subcooled liquid nitrogen, called "slush", which is at a temperature of -210 ° C, lower than liquid nitrogen, and where you can reduce the Leidenfrost effect (formation of a layer of steam around the sample that prevents efficient heat transfer). The introduction of the cell containers previously named in slush means a cooling rate of 10,000 ° C / min or about 20,000 ° C / min (depending on the author, [ MA Nowshari and G. Bren, Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos, Human Reproduction 15 (2001), 2368-2373]; [G. Vajta, P. Holm, T. Greve and H. Callesen, Vitrification of porcine embryos using the Open Pulled Straw (OPS) method, Acta Vet. Scand. 38 (1997) 349-352 ]) for PAHO, and similar results in the case of cryoloops [ T. Mukaida, S. Nakamura, T. Tomiyama, S. Wada, M. Kasai and K. Takahashi, Succesful birth alter transfer of vitrified human blastocysts with us of a cryoloop containerless technique, Fertil. Steril 76 (2001) 618-620] or copper gratings [PL Steponkus, SP Myers and DV Lynch, et al , Cryopreservation of Drosophila melanogaster embryos, Nature 345 (1990) 170-172 ], results that, although interesting, are still not enough to achieve adequate vitrification for many cell types.

The overheating process is even more troubled. All overheating processes are based on the transfer of the cell container from liquid nitrogen (where the sample has been kept indefinitely) up to a container container of a reheat solution (normally at 37.5º), passing through the air between the two containers This passage through the air is a serious inconvenience, because during this step the overheating takes place at a very slow speed, in which it is impossible to avoid training and ice crystal growth

The probability of adequate vitrification depends, finally, on the volume of the sample. Minimizing the volume of the sample minimizes the amount of liquid susceptible to nucleation, and therefore increases the chances of proper vitrification. With this philosophy, also closely related to the maximization of the cooling rate discussed above, the "minimum volume techniques" were born, such as Cryotop [ Kuwayama M, Vajta G, Kato O, Leibo S. P, Highly efficient vitrification method for cryopreservation of human oocytes, Reprod Biomed Online, 2005; 11; 300-308 ] or, more recently, CryoTip [ Kuwayama M, Vajta G, lEda S, Kato O, Vitrification of human embryos using the CryoTip method, Reprod Biomed Online, in press ], which have reaped some successes in vitrification of complicated cell types of cryopreservation. However, the difficulty in handling the sample in such containers (in the case of Cryotop), or the need for higher cooling rates (12,000 ° C / min in the case of CryoTip, and slightly higher in the case of Cryotop) make The invention of new procedures is necessary.

Description of the figures

Figure 1. Experimental setup for the Development of the experiments. The liquid nitrogen contained in the cylindrical container is set in motion by a vertical stirrer with the blade located about 2 cm from the edge lower. The speed of rotation of this agitator could be controlled between 0 and 2700 rpm (measured with a Hall effect sensor and an oscilloscope)

Figure 2. A). Quartz capillary cooling when introduced in liquid nitrogen, stirred at 2700 rpm. B) OPS capillary cooling when introduced into nitrogen liquid, without forced convection.

Figure 3. Increase the speed of cooling with stirring speed.

Description of the invention

The process object of the present invention it comes to solve the previously mentioned inconveniences, maintaining the advantages of straw type containers and getting cooling speeds higher than any other technique, avoiding the nucleation and growth of ice in the vitrification process, with the consequent reduction in toxicity generated by the use of high concentrations of cryoprotectant

The procedure deals with the application to concrete problem of cellular cryopreservation of a technique used Widely in engineering to increase heat transfer (forced convection), which is complemented by a new cellular container, quartz capillaries (frequently used for X-ray diffraction), and the use of cryogenic mixtures alternatives to liquid nitrogen, such as slush (liquid nitrogen subcooled from the phase change of liquid nitrogen) or slurry (mixture of liquid nitrogen with different particles such as copper powder or sodium chloride, depending on the characteristics of the sample to be cryopreserved). In the process of overheating makes use of the physical properties of the change phase slush to liquid nitrogen, also complemented with the forced convection technique with the reheating fluid, with the same objective of achieving a high transfer of hot.

Forced convection occurs as consequence of the relative movement of the quartz capillary with with respect to the cooling (or reheating) fluid. Can be implemented in many ways: capillary movement, movement of the cooling (or reheating) fluid by means of a narrowing of the flow, subject to a pressure gradient, by agitation, etc. The technically easiest way to implement forced and more easily repeatable convection is by stirring the coolant or reheating fluid, by what this has been the form chosen for the realization of the Experiments set forth in the subsequent examples.

The cooling process requires first place of choice of cell container and fluid refrigerant in which it will be introduced. As a cellular container they use quartz capillaries, and as a coolant they can be used Liquid nitrogen, slush or slurry.

The use of quartz capillaries to get high cooling and reheating speeds are motivated due to the great thermal conductivity that quartz has (about 6.5 W m <-1> K <-1>, compared with the PVC of the OPS capillaries, about 0.19 W m -1 K -1), as well as the small diameter inside (about 0.100 mm - 0.200 mm) and reduced dimensions of the wall that can be achieved with the state Current technique for this material (about 0.010 mm). Others materials could be equally used, such as silver or other high thermal conductivity metals, as long as the dimensions were similar to those of quartz capillaries commented here.

As a cooling fluid, you can choose between Liquid nitrogen, slush or slurry.

The appropriate security measures must be taken for the management of the cryogenic mixture.

The way to obtain slush (subcooled liquid nitrogen) is by vacuum. A simple vacuum pump achieves solidification of part of the liquid nitrogen in a few minutes (another part is lost due to evaporation). Upon returning to normal atmospheric pressure, the nitrogen collapses and a subcooled liquid nitrogen is formed, with some solid particles in it, which is called " slush ".

If the coolant chosen is slurry, the volume of the particles chosen will be about three quarters parts of the total volume (the rest is filled with nitrogen liquid), this volume depending on the nature of the particle chosen The chosen particles can be of a great variety. Be they have obtained good results with powdered copper particles and with sodium chloride. It is necessary to homogenize the slurry to get a good mix between liquid nitrogen and particles contained in it.

The cooling process itself It consists of the introduction of the quartz capillary, container of the cells that we want to cryopreserve, within a flow of cryogenic fluid chosen from the options already mentioned (liquid nitrogen, slush or slurry). The objective is to increase the cooling rate of the cells contained in the capillary to achieve adequate vitrification with low concentrations Cryoprotectant Forced convection occurs as consequence of the relative movement of the quartz capillary with regarding the cryogenic fluid. It has a double advantage over to simple immersion:

one)

Replacing fluid layers refrigerant heated by the introduction of the sample by others colder layers and consequently with greater capacity refrigerant.

2)

Destabilization and / or elimination of Leidenfrost vapor layer, if formed, which prevents a good thermal contact between the cell container and the fluid refrigerant.

Forced convection can be implemented in many shapes, capillary movement, fluid movement refrigerant by narrowing the flow, by agitation, etc. The technically easiest way to implement the forced and more easily repeatable convection is by agitation of the cooling fluid, so this has been the chosen form for carrying out the experiments exposed in the examples.

Once the sample has reached its temperature minimum, the capillary must be moved, along with a reservoir filled with liquid nitrogen, to the final nitrogen container liquid, where it will remain for the entire duration of the cryopreservation

The cryopreservation procedure is completed with the reheating process, which is also about of increasing heat transfer. First, use is made of the physical properties of the phase change from slush to nitrogen liquid to avoid the inconvenience of the passage of the container cell by air that separates liquid nitrogen containers and overheating solution. For this, the liquid nitrogen where the sample has been vitrified cell by liquid nitrogen slush. There is no problem in performing said substitution since the liquid nitrogen slush has a higher density and liquid nitrogen evaporates quickly. He slush, during the time that has elapsed since the quartz capillary of it until it is introduced into the solution of overheating, keeps the sample temperature constant cell at -196 ° C, as a result of the change in state of said slush, attached to the quartz capillary, to liquid nitrogen.

This temperature maintenance, get prevent icing (the vitrified sample is so viscose that at -196 ° C still cannot form ice crystals) and allows the temperature of the cell sample to increase by abrupt and sudden form when introduced into the solution of overheating

The reheating process is completed finally with the use of forced convection, in a solution of reheating with suitable fluids, such as water at 37.5 ° C or others fluids with greater thermal diffusivity, to which it can be added copper powder particles to increase conduction thermal

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Embodiment of the invention Example 1 Cooling speed measure

Cellular container: Quartz capillaries (Capillary Tube Supplies Ltd. UK), with the following features:

Inner diameter 0.200 mm.

0.010 mm wall thickness.

Capillary length: 90 mm.

Coolant: liquid nitrogen. This coolant is poured into an insulating container (which minimize evaporation of liquid nitrogen). The volume of coolant existing in said container must be the high enough to cover most of the capillary Quartz being introduced.

Forced convection procedure used: agitation of the cooling fluid (liquid nitrogen) in the anterior vessel using a vertical stirrer that rotates on its axis of symmetry (see figure 1) at a rotation speed of 2700 rpm

Capillary nitrogen entry rate liquid: 1250 mm / s (measured with the photoelectric barrier device of fork with counter, Phywe 11207.30).

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Results

Cooling speed: 122,500ºC / minute (between –10ºC and -170ºC).

The measurement was made with a type T thermocouple (Omega Engineering Inc), diameter 0.025 mm, introduced in the capillary with the union 0.5 cm from the lower end. The card of acquisition (Measurement Computing, USB-1208LS) Sampled at 600 samples per second.

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Conclusions

This cooling rate achieved (in the Figure 2A shows the cooling profile of this experiment) greatly exceeds the cooling rates achieved by the rest of methods. (As an example, Figure 2B shows the cooling profile for a conventional OPS capillary introduced into liquid nitrogen without a convection process forced, measured with another Omega type T thermocouple Engineering Inc., of measures coinciding with the previous and the same acquisition card).

Figure 3 shows the cooling rate of quartz capillaries to be introduced in liquid nitrogen in the setup shown in the figure 1 with different stirring speeds. These results suggest that the agitation speed of the coolant during the introduction of the quartz capillary containing this material cell phone, should be as high as possible and allow technical means.

This cooling rate is greater than the achieved with no other technique used so far, and allows lower concentrations of cryoprotectant, and, for consequently, less toxic than those used so far.

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Example 2 Cell preservation procedure

Cellular container: Quartz capillaries (Capillary Tube Supplies Ltd. UK), with the following features:

Inner diameter 0.200 mm.

0.010 mm wall thickness.

Cells to cryopreserve: mouse fibroblasts (3Q3). The culture medium used is DMEM with 10% Serum Fetal Bovine and 2% Penicillin and Streptomycin. Incubation it is carried out in an atmosphere with 5% CO2 at 37ºC and in saturation humidity conditions.

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Cryoprotective solution: 1.5 M of 1.2 propanediol and 0.3 M sucrose in PBS (buffer solution that keeps the PH constant).

Coolant: slush. The procedure of slush manufacturing was by means of a vacuum pump (Telstar 2F3), which converted a volume of 390 ml of liquid nitrogen into slush in 34 minutes. The slush was poured into the same container than in figure 1, with the same volume as shown in the Figure 1.

Forced convection procedure used: stirring of the cooling fluid (slush) in the same container anterior by a vertical stirrer that rotates on its axis of symmetry (as in the previous example, see figure 1) to a 500 rpm rotation speed.

Capillary nitrogen entry rate slush liquid: 1250 mm / s (measured with the barrier device photoelectric fork with counter, Phywe 11207.30).

Overheating: Use of physical properties of the phase change of the slush attached to the quartz capillary to liquid nitrogen. Immersion of the capillary in a 37.5ºC water bath no forced convection

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Results

Cooling speed: 158,000ºC / minute (measured with thermocouple type T Omega Engineering Inc, diameter 0.025 mm and Measurement Computing acquisition card, USB-1208LS at 600 samples / s).

Survival of fibroblasts: 95% (technique trypan blue).

When the chosen coolant is slush, the vacuum pump must work long enough to get a good slush quality (liquid nitrogen must be solidified), but less time than it produces so much evaporation of liquid nitrogen so that the resulting slush do not cover most of the quartz capillary being this one introduced in the container container.

Claims (3)

1. Cellular cryopreservation by vitrification with low concentrations of cryoprotectant characterized in that it consists of the introduction of the cellular container to be cryopreserved in a refrigerant fluid to increase the cooling rate of the cells and subsequent reheating by forced convection. 2. Cellular cryopreservation by vitrification with low concentrations of cryoprotectant according to claim 1, characterized in that the cellular container is a quartz capillary and the cooling fluid is liquid nitrogen, slush or slurry. 3. Cellular cryopreservation by vitrification with low concentrations of cryoprotectant according to previous claims, characterized in that the forced convection is produced by movement of the quartz capillary with respect to the cooling fluid.