Method of freezing making use of a mineral nucleator

Abstract

The present invention relates to a method for freezing a water-containing quantity of a biological entity or a formulation in a vessel using a mineral nucleator, to the use of the mineral as a nucleator and to a vessel with the mineral in or on the whole or part of a surface thereof.

Claims

1. A method for freezing a water-containing quantity of a biological entity or a formulation comprising: contacting the water-containing quantity of the biological entity or formulation with a framework silicate in a vessel to form a solution; and freezing the solution at a supercooling temperature between 0 C. and 8 C. in the vessel, wherein the framework silicate promotes the non-spontaneous formation of ice at a supercooling temperature between 0 C. and 8 C.

2. A method as claimed in claim 1 wherein the framework silicate is selected from the group consisting of Feldspar, Silica, Nepheline, Petalite, Leucite, Sodalite, Cancrinite, Scapolite, Analcite and Zeolite.

3. A method as claimed in claim 1 wherein the framework silicate is a framework aluminosilicate.

4. A method as claimed in claim 1 wherein the framework silicate is a Feldspar or Feldsapthoid.

5. A method as claimed in claim 1 wherein the framework silicate is a Feldspar.

6. A method as claimed in claim 1 wherein the framework silicate is a Feldspar with a predominance of NaAlSi.sub.3O.sub.8 and KAlSi.sub.3O.sub.8.

7. A method as claimed in claim 1 wherein the framework silicate is a Feldspar with a predominance of KAlSi.sub.3O.sub.8.

8. A method as claimed in claim 1 wherein the framework silicate is a Feldspar with a predominance of CaAl.sub.2Si.sub.2O.sub.8 and NaAlSi.sub.3O.sub.8.

9. A method as claimed in claim 1 wherein the framework silicate is a Feldspar with a predominance of NaAlSi.sub.3O.sub.8.

10. A method as claimed in claim 1 wherein the water-containing quantity is a water-containing quantity of a medical formulation.

11. A method as claimed in claim 1 wherein the water-containing quantity is a water-containing quantity of a formulated foodstuff.

12. A method as claimed in claim 1 wherein the water-containing quantity is a water-containing quantity of a biological entity.

13. A method as claimed in claim 12 wherein the biological entity is a cell or aggregate of cells.

14. A method as claimed in claim 1 wherein the contacting the water-containing quantity with a framework silicate in a vessel comprises: adding the water-containing quantity to the vessel; and adding the framework silicate in a discrete form to the vessel.

15. A method as claimed in claim 1 wherein the framework silicate is in or on the whole or part of a surface of the vessel or part thereof such that active nucleation sites of the framework silicate are exposed effectively to the water-containing quantity and the contacting the water-containing quantity with a framework silicate in a vessel comprises adding the water-containing quantity to the vessel.

16. A method for freezing a water-containing quantity of a biological entity or a formulation comprising: forming a solution comprising the water-containing quantity of the biological entity or formulation, and a framework silicate; freezing the solution at a supercooling temperature between 0 C. and 8 C.; and during the freezing, promoting the non-spontaneous formation of ice at the supercooling temperature between 0 C. and 8 C.

17. The method of claim 16 wherein the framework silicate comprises one or more of Feldspar, Silica, Nepheline, Petalite, Leucite, Sodalite, Cancrinite, Scapolite, Analcite and/or Zeolite.

18. The method of claim 16 wherein the forming the solution comprises: adding the water-containing quantity to a vessel; and adding the framework silicate in a discrete form to the vessel.

Description

(1) Various embodiments of the invention will now be described in a non-limitative sense only with reference to the following Examples and Figures in which:

(2) FIG. 1 shows the fraction of straws filled with pure water frozen as a function of temperature with and without the nucleator BCS-CRM 376/1 Potash Feldspar during a 1 C./min cooling ramp (diamonds are straws with Feldspar and squares are those without);

(3) FIG. 2 shows the fraction of straws filled with 10% v/v ethylene glycol frozen as a function of temperature with and without the nucleator BCS-CRM 376/1 Potash Feldspar during a 1 C./min cooling ramp (diamonds are straws with Feldspar and squares are those without. The black line indicates the melting temperature of the liquid frozen);

(4) FIG. 3 shows viability data for multicellular liver spheroids encapsulated in alginate after cryopreservation was carried out using a slow-cooling method (a) without nucleator, (b) with cholesterol as a standard nucleator, (c) with Feldspar as a dust (1 m to 5 m diameter) and (d) with Feldspar as a bead (6 mm diameter); and

(5) FIG. 4 shows (a) ice cream treated with SnoMax, b) untreated ice cream, c) ice cream treated with K-Feldspar and d) a micrometer at the same magnification as the three samples. The small graduations are 10 m apart.

EXAMPLE 1NUCLEATION EXPERIMENTS IN CRYOVIALS

(6) Experiments were carried out in cryovials (Thermo Fisher) placed in an MF2000 prototype electrically powered controlled rate freezer (Asymptote Ltd, Cambridge, UK). The vials were cooled from room temperature down to 4 C. at a rate of 2 C. per minute, then held at 4 C. for 5 minutes and then cooled at 1 C. per minute until nucleation occurred. Nucleation was registered by a T-type thermocouple in each vial and noting the temperature at which the heat of fusion was released. Table 1 shows the average nucleating temperatures of a range of Feldspar materials placed in different solutions of water.

(7) TABLE-US-00001 TABLE 1 Average Standard temperature deviation of of nucleation nucleation, temperature, Nucleator Liquid C. C. None 10% glycerol 9.74 1.10 (thermocouples) in water One or two grains of 10% glycerol 7.17 2.10 Forshammer Feldspar in water in each vial (average weight 0.04 grammes) 0.060% wt BCS-CRM 10% glycerol 7.02 0.96 376/1 Potash Feldspar in water 0.030% wt BCS-CRM 10% glycerol 6.93 1.24 376/1 Potash Feldspar in water None De-ionised 9.08 1.98 (thermocouples) water 0.030% wt BCS-CRM De-ionised 7.24 1.96 376/1 Potash Feldspar water 0.060% wt BCS-CRM De-ionised 6.27 1.58 376/1 Potash Feldspar water One or two grains of De-ionised 5.71 2.48 Forshammer Feldspar water in each vial (average weight 0.04 grammes) One bead of feldspar De-ionised 4.40 1.44 in each vial, 6 mm water diameter

EXAMPLE 2NUCLEATION EXPERIMENTS IN STRAWS

(8) Experiments were carried out in straws for cryopreservation of sperm cells. The straws contained PVA (polyvinyl acetate) in one end enclosed between two cotton plugs. The PVA was emptied out from the straw and replaced with the mineral nucleator in powder form. The straws were placed in an EF600 electrically powered controlled rate freezer (Asymptote Ltd, Cambridge, UK) and cooled from room temperature down to 4 C. at a rate of 2 C. per minute, then held at 4 C. for 5 minutes and then cooled at 1 C. per minute until nucleation occurred. Nucleation was registered by a T-type thermocouple in each straw at the opposite end to the nucleator and noting the temperature at which the heat of fusion was released.

(9) FIG. 1 shows the fraction of straws filled with pure water frozen as a function of temperature with and without the nucleator BCS-CRM 376/1 Potash Feldspar during the 1 C./min cooling ramp (blue diamonds are straws with feldspar and red squares are those without). In this case the freezing temperature was determined by calibrating the relationship between the internal temperature of the straws and the indicated temperature of the EF600 freezer and recording the temperature indicated at the point of freezing. Table 2 shows the average nucleating temperatures with and without the stated nucleators.

(10) TABLE-US-00002 TABLE 2 Standard Average deviation temperature of of nucleation nucleation, temperature, Nucleator Liquid C. C. None De-ionised 9.21 1.77 (thermocouples) water BCS-CRM 376/1 De-ionised 6.09 1.94 Potash Feldspar water Silica 22 De-ionised 7.41 1.17 water

(11) The experiment was repeated for ethylene glycol in place of pure water. FIG. 2 shows the fraction of straws filled with 10% v/v ethylene glycol frozen as a function of temperature with and without the nucleator BCS-CRM 376/1 Potash Feldspar during the 1 C./min cooling ramp (blue diamonds are straws with Feldspar and red squares are those without). The black line indicates the melting temperature of the liquid frozen. Freezing temperature was determined as described above.

EXAMPLE 3STABILITY IN SOLUTION

(12) Different preparations of Feldspar were prepared and tested as in Example 1 then left in a consumer grade refrigerator for two weeks and finally tested again using the same experimental protocol. Table 3 shows the average nucleation temperatures for first and second runs of the different Feldspar solutions.

(13) TABLE-US-00003 TABLE 3 Average Average temperature temperature of of nucleation nucleation C., C., first second Nucleator Liquid run run 0.060% wt BCS- De-ionised 6.27 7.65 CRM 376/1 water Potash Feldspar 0.030% wt BCS- De-ionised 7.24 8.21 CRM 376/1 water Potash Feldspar 0.060% wt BCS- 10% glycerol 7.02 6.88 CRM 376/1 in water Potash Feldspar 0.030% wt BCS- 10% glycerol 6.93 7.42 CRM 376/1 in water Potash Feldspar

EXAMPLE 4VIABILITY DATA

(14) An experiment was carried out 72 hours post thaw to determine recovery of multicellular liver spheroids encapsulated in alginate. Viable cell numbers were measured using metabolic vital dyes (flourescein diacetate and propidium iodide) Cryopreservation was carried out using a slow-cooling method with 12% DMSO as cryoprotectant. Samples were cooled (a) without nucleator, (b) with cholesterol as a standard nucleator, (c) with Feldspar as a dust (1 m to 5 m diameter) and (d) with feldspar as a bead (6 mm diameter). The results are shown in FIG. 3.

EXAMPLE 5ICE CREAM CRYSTAL SIZES

(15) To demonstrate the effectiveness of a mineral powder for reducing the size of crystals in liquid food products, experiments were performed using a home ice cream maker and commercially available vanilla Haagen-Daz ice cream. Experiments were conducted using 500 ml pure melted ice cream, 500 ml melted ice cream mixed with 3 grains of dissolved Snomax and 500 ml melted ice cream mixed with 1.2 g of powdered K-Feldspar dispersed in 10 ml of water. All three samples were placed in the ice cream maker at a starting temperature of approximately 15 C. and the ice cream maker switched on. The ice cream maker cooled the ice cream down to approximately 20 C. under constant stirring. The ice cream was then quickly transferred to a plastic vessel and placed in a freezer at 18 C. where it was left for 36 hours. At all points great care was taken to prevent cross contamination of ice cream samples.

(16) After this 36 hour period the ice cream was recovered from the freezer and a small amount placed between two microscope slides precooled to 35 C. The slides were then placed onto a transmission microscope equipped with a liquid nitrogen cooled coldstage. FIG. 4 shows typical pictures of the ice crystals in each ice cream sample. The mean size of ice crystals formed in untreated ice cream was determined to be 2711 m. In ice cream treated with Snomax the mean size was found to be 123 m and in ice cream treated with K-Feldspar the mean size was found to be 145 m.