KR20130069378A - Cryopreservation technique using antifreeze protein from leucosporidium sp - Google Patents

Abstract

PURPOSE: An AFP(Anti-Freeze Protein) derived from Leucosporidium sp. is provided to enhance viability when freezing reproductive cells or biological tissues, and to be used as a composition for cryopreservation of the reproductive cells or biological tissues. CONSTITUTION: A composition for cryopreservation of reproductive cells or biological tissues contains an AFP derived from Leucosporidium sp. The AFP protein has an amino acid sequence of sequence number 1. A method for cryopreservation of reproductive cells or biological tissues using the AFP comprises: a step of collecting semen from animals exclusive of human; a step of diluting the semen using diluents; a step of centrifuging the diluted semen and removing supernatant to obtain a sperm precipitate; and a step of diluting the sperm precipitate, adding AFP, and freezing.

Description

[0001] The present invention relates to a cryopreservation technique using antifreeze protein from Leucosporidium sp.

The present invention relates to a method for cryopreservation of germ cells or living tissues using Antifreeze protein (AFP) derived from a microorganism of the genus Lucosporium.

Anti-freeze proteins (AFP), called ice-binding proteins, are synthesized in polar and cold organisms (Yu and Lu (2011) PLoS One 6: e20445). AFP is a protein that binds to ice to inhibit the growth of ice crystals, allowing organisms to overcome cold weather and survive. This protein and ice are bound through hydrogen bonds. AFP-unbonded ice can damage cells, but AFP-bound ice does not produce ice crystals that can damage cells, allowing organisms to survive at extreme low temperatures. Therefore, there is a large difference between AFP-bound ice cells and non-AFP-bound ice cells (Garnham et al. (2011) Proc Natl Acad Sci U SA 108: 7363-7367). Generally, ice undergoes a recrystallization process in which cells are damaged by the large ice crystals formed. The main role of AFP is to inhibit crystal formation and recrystallization of ice to reduce damage to cells to be frozen (Rubinsky et al. (2010) Neurosci Res 67: 256-259). Since AFP inhibits the formation of ice crystals, there is a difference between the freezing point and the melting point of water. This phenomenon is called thermal hysteresis activity and is a quantitative indicator of the activity of AFP. Thermal history can be measured with a device called a nanoliter osmometer (Kristiansen et al. (2011) Insect Biochem Mol Biol 41: 109-117).

On the other hand, the cryopreservation technique has become necessary as embryo technology such as in vitro fertilization, replication, and transformation is developed. However, various cryopreservation technologies developed so far have caused serious morphological and biochemical changes leading to collapse of intracellular organ, intracellular skeletal injury, cell death, and reduced viability of embryos.

As a result of efforts made to solve the problems of the prior art as described above, the present inventors have found that, in the case of Leucosporidium The present invention has been accomplished by confirming that the viability of a living cell or a living tissue is enhanced when a living cell or a living tissue is frozen using an AFP protein derived from a microorganism.

It is an object of the present invention to provide a composition for cryopreservation of germ cells or living tissues comprising an AFP protein derived from a microorganism belonging to the genus Luciferidium.

It is still another object of the present invention to provide a method for cryopreserving germ cells or living tissues using AFP protein derived from microorganism of the genus Lucoidarium.

In order to accomplish the above object, the present invention provides a composition for cryopreservation of germ cells or living tissues comprising an AFP protein derived from a microorganism belonging to the genus Luciferidium.

The present invention also provides a method for cryopreserving germ cells or living tissues using an AFP protein derived from a microorganism of the genus Luciferidium.

The AFP protein derived from Luciferidium genus microorganism according to the present invention has an excellent effect of enhancing the viability of germ cells or living tissues upon freezing, and thus can be usefully used as a composition for cryopreservation of germ cells or living tissues.

Figure 1 shows the degree of DNA damage of spermatozoa thawed by Comet assay (left upper: AFP 0 μg / ml; right upper: AFP 0.01 μg / ml; lower left: AFP 0.1 μg / ml; / ml).
FIG. 2 is a graph showing damage of plasma membranes of sperm thawed using SYBR14 / PI fluorescence staining.
FIG. 3 is a graph showing damage of plasma membrane of sperm thawed using FITC-PNA / PI fluorescence staining.
FIG. 4 shows H & E staining results of heart valve tissue stored for 24 hours in normal heart valve tissues and preservatives (left: normal heart valve, right: heart valves stored in a preservative for 24 hours).
FIG. 5 is a TUNEL staining result of heart valve tissue stored for 24 hours in normal heart valve tissue and preservative (left: normal heart valve, right: heart valve stored in a preservative for 24 hours).

The present invention provides a composition for cryopreserving a germ cell or a living tissue comprising an anti-freeze protein (AFP) derived from a microorganism belonging to the genus Lucosporium.

Hereinafter, the present invention will be described in detail.

The AOP protein derived from Luciferidium sp. Microorganism of the present invention was collected from a lake ice of the Arctic Svalbard archipelago, and then purified from the plate medium and purified by ITS1 (ribosomal internal transcribed spacer 1), the result of ITS2 and 5.8S gene analysis (Connell et al. (2008) Microb Ecol 56, 448-459), preferably derived from a yeast designated Luocoidolium AY30, 1 < / RTI > and functional equivalents of said protein. Is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70%, more preferably at least 90%, more preferably at least 90% Refers to a protein having a homology of at least 95% and exhibiting a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 1.

The AFP protein of the present invention includes not only a protein having a native amino acid sequence thereof but also an amino acid sequence variant thereof, within the scope of the present invention. A variant of an AFP protein refers to a protein having a sequence that differs by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, with an AFP native amino acid sequence and one or more amino acid residues. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In some cases, it may be modified by phosphorylation, sulfation, acetylation, glycosylation, methylation, farnesylation, or the like.

The composition for cryopreservation may further comprise a medium of Modena B as a diluent and is preferably selected from the group consisting of glucose, EDTA, sodium citrate, sodium hydrogencarbonate, tris, citric acid, cysteine, BSA, kanamycin sulfate, The modified Modena B medium may also be included as a diluent.

In the present invention, germ cells include gamete, that is, sperm or striatum of an oocyte, including, but not limited to, primordial germ cell, embryonic germ cell, A testicular germ cell, a spermatogonial stem cell, and a germinal stem cell, preferably a sperm. In addition, the biotissue in the present invention is not limited to its origin.

The AFP protein derived from micrococcus of Luciferidium according to the present invention has an excellent effect of enhancing the viability of a germ cell or a living tissue upon freezing.

In addition,

(a) collecting semen from an animal other than a human;

(b) diluting the harvested semen using a diluent;

(c) centrifuging the diluted semen to remove supernatant and obtaining a sperm deposit; And

(d) diluting the spermatogonial precipitate, adding an anti-freeze protein (AFP) derived from a microorganism belonging to the genus Lucoidium, and cooling to obtain a frozen spermatozoon. A method for cryopreserving a germ cell or a living tissue using the same.

The diluent of step (b) is not limited thereto, but it is preferably composed of a modified Modena B medium, a LEY diluent and a LEYGO diluent, which are basic diluents, a cold diluent and a freezing diluent.

The modified Modena B medium is preferably, but not limited to, glucose, EDTA, sodium citrate, sodium hydrogencarbonate, tris, citric acid, cysteine, BSA and kanamycin sulfate.

The cooling in the step (d) is not limited to this, but it is preferable to perform a primary cooling with liquid nitrogen vapor and a secondary cooling with liquid nitrogen for safe freezing of germ cells or living tissues.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited to the following examples.

Example  1. Semen collection and freezing

Sperm semen (Kangwon Univ.) Was collected in a 50 mL plastic centrifuge tube containing a large amount of sperm. The collected sperm was transported in a sealed state in an ice box to prevent rapid temperature changes.

Modena B, 20 ml: Glucose 6 g, EDTA 0.45, and LEYGO extender were used as a diluent, a modified diluent, a modified diluent, and a LEY extender, respectively, to dilute the semen of the mini-pig. (80%) of 310 mM b-lactose (v / v), 1 g of sodium citrate, 0.2 g of sodium bicarbonate, 1 g of Tris, 0.5 g of citric acid, 0.01 g of cysteine, 0.8 g of BSA, 0.06 g of kanamycin sulfate, ), 20% Egg yolk (v / v), Kanamycin sulfate (100 mg / ml)) and LEYGO extender (89.5% LEY extender (v / v), 9% Glycerol v), 100 mM Trehalose (w / v), various concentration of AFP 0 mM, 0.1 mM, 0.01 mM, 0.001 mM). The semen was diluted using the diluent, and the supernatant was removed by centrifugation at 17O < 0 > C and 2040 RPM for 10 minutes. The resulting spermatozoa were diluted again with 50 ml of modified Modena B, centrifuged to remove the supernatant, diluted with 20 ml of modified Modena B, and centrifuged to remove supernatant. The final spermatozoa precipitate was diluted with a LEY extender to a concentration of 1.5x10 ⁹ sperm / ml, shaken carefully and allowed to stand for 1 hour in ice water to reduce damage due to rapid temperature changes. Then, a LEYGO extender containing AFP-derived AFP30 protein was added to give a final concentration of 1.0 x ^{10 9} sperm / ml, followed by primary cooling with liquid nitrogen vapor and secondary cooling with liquid nitrogen.

Experimental Example  1. Measurement of motility of thawed sperm

In order to confirm the effect of the addition of the AFP protein derived from Luciosporiidium microorganism on frozen cells in Example 1, the frozen sperm was thawed to measure the motility of the sperm. First, the temperature of the hot water tank was adjusted to 50 ° C and the temperature of the modified Modena B was adjusted to 37 ° C. The straw containing frozen sperm was dissolved in a hot water bath at 50 ° C for 5 seconds and then diluted in modified Modena B at 37 ° C . This was washed by centrifugation to remove supernatant, and 3 mL was dropped on a standard count chamber slide of 20 micron thickness. This was observed through a 100x microscope and statistical data were obtained by observing more than 10 sites through HELOS (Hamilton Thorne, Beverly, Mass., USA). The sperm motility was measured using a computer-assisted analysis of sperm motility (CASA) using curve speed (VCL), linear velocity (VSL), mean velocity (VAP), linearity coefficient (LIN), density (DANCE) And so on. The results are shown in Table 1.

Classification Motility (%) Progressive (%) VAP (μm / s) VSL (μm / s) VCL (μm / s) ALH (μm) Fresh 80.4 ± 2.5 ^c 71.2 ± 2.2 ^c 54.48 ± 1.88 ^c 33.84 ± 1.15 ^bc 100.62 + 4.67 ^a 6.20 ± 0.23 ^b Control 68.2 + 2.4 ^a 61.7 ± 2.3 ^a 64.97 + - 3.25 ^b 42.97 ± 2.69 ^a 100.78 + - 2.38 ^a 5.62 ± 0.30 ^a AFP (0.01 mg / ml) 74.6 ± 2.6 ^d 68.0 ± 3.2 ^c 58.16 ± 1.51 ^ac 33.78 ± 0.75 ^bc 99.92 + 3.51 ^a 6.40 ± 0.21 ^b AFP (0.1 mg / ml) 67.8 ± 3.9 ^a 61.4 + 4.4 ^a 60.86 ± 5.51 ^ab 37.44 ± 6.23 ^ab 99.58 + 4.92 ^a 6.08 ± 0.35 ^ab AFP (1 mg / ml) 47.0 ± 1.6 ^b 40.6 + 1.7 ^b 52.42 ± 1.06 ^c 28.06 + - 0.67 ^c 91.54 ± 3.20 ^b 6.42 + 0.24 ^b p- Value p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.01 p < 0.001

As shown in Table 1, it was confirmed that the mobility of thawed sperm was greatly increased when the microorganism-derived AFP protein derived from Luciferidium was added to the medium and frozen.

Experimental Example  2. Of thawed sperm DNA  Measure damage

In order to confirm the effect of the addition of the AFP protein derived from Luciferidium genus microorganism on the DNA damage of the frozen cells in Example 1, thawed spermatozoa in the same manner as in Experimental Example 1 was subjected to Comet assay Respectively. More specifically, the thawed sperm samples obtained in Experimental Example 1 were cultured in a 2% 1-mercaptoethanol solution at 4 ° C for 2 hours and centrifuged (400 x g , 5 minutes) to obtain Ca ^{2 +} , And resuspended in Mg ^{2 +} -free PBS (phosphate buffered saline). The sperm samples were diluted with 0.7 agarose and coated on slides. The slides were then immersed in CometAssay Lysis solution (Trevigen, Maryland, USA) for 2 hours at 4 ° C and then incubated in RNase and proteinase K for 4 hours To remove RNA and protein. This was treated with an alkaline solution (NaOH, 200 mM EDTA; pH 13) for 30 minutes, and the dissolved slides were then electrophoresed at 12 V, 300 mA for 1 hour. After that, the cells were reacted with SYBR green fluorescent dye for 5 minutes and observed with a fluorescence microscope. The results are shown in FIG. 1 and Table 2.

Classification Tail DNA (%) Tail Length (μm) Olive Moment Control 7.1 ± 8.1 ^ab 2.2 ± 2.4 0.75 ± 0.87 ^ab AFP (0.01 mg / ml) 6.5 ± 7.5 ^a 1.9 ± 1.5 0.59 + 0.63 ^a AFP (0.1 mg / ml) 11.6 ± 7.5 ^ab 2.5 ± 1.4 1.04 ± 0.72 ^ab AFP (1 mg / ml) 13.4 ± 9.6 ^b 3.0 ± 1.8 1.29 ± 0.92 ^b p- Value  0.0235 0.324 0.0313

As shown in Fig. 1 and Table 2, when the AFP protein derived from Luciferidium genus microorganism was added to the medium and cryopreserved, the degree of DNA damage of the thawed sperm was greatly reduced.

Experimental Example  3. Measurement of plasma membrane damage of thawed spermatozoa

The following experiment was conducted to confirm the effect of addition of AFP protein derived from micrococcus of Luciferidium genus on plasma membrane damage of frozen cells in Example 1 above. First, the thawed sperm sample obtained in Example 1 was diluted with a BTS extender and adjusted to a concentration of 1 x 10 ⁶ spermatozoa / mL. The sperm samples were dispensed in 500 μl aliquots, and 100 nM and 12 μM of SYBR14 and PI, respectively, were added to give final concentrations. Flow cytometry was performed using a FC 500 series cytometer (Beckman Coulter, Inc., Miami, FL, USA) and flow cytometry was analyzed using the CXP program (Beckman Coulter, Inc.). The results are shown in Figure 2 and Table 3.

Classification Dead (PI + SYBR-) Dying (PI + SYBR +) Live (PI-SYBR +) Control 51.9 ± 5.5 ^a 9.3 ± 5.0 ^ab 38.8 ± 0.8 ^c AFP (0.01 mg / ml) 45.2 ± 7.6 ^a 18.4 ± 7.3 ^a 36.4 ± 0.9 ^d AFP (0.1 mg / ml) 47.8 ± 19.0 ^a 18.8 ± 19.3 ^a 33.4 ± 0.7 ^a AFP (1 mg / ml) 72.0 ± 0.9 ^b 0.46 ± 0.1 ^b 27.5 ± 0.9 ^b p- Value p < 0.001 p < 0.001 p < 0.001

As shown in FIG. 2 and Table 3, when the AFP protein derived from Luciferidium genus microorganism was added to the medium and cryopreserved, it was confirmed that the degree of damage of the plasma membranes of the thawed sperm was greatly reduced.

Experimental Example  4. Of thawed sperm Acrosome  Measure damage

Fluorescein isothiocyanate (FITC-PNA; Lectin from Arachis hypogaea, Sigma-Aldrich, St. Louis, MO) was used to confirm the effect of the addition of the AFP protein derived from Luciferidium genus microorganism on the acrosome- , MO, USA) / PI fluorescence staining technique was performed. First, the thawed sperm samples obtained in Example 1 were dispensed in a volume of 500 μl at a concentration of 1 × 10 ⁶ spermatozoa / mL, 10 μl of FITC-PNA (50 μg / ml) and PI (750 μM) &Lt; / RTI &gt; for 5 minutes, followed by flow cytometry. The results are shown in FIG. 3 and Table 4.

Classification Acrosomal membrane PI + / PNA- (%) PI + / PNA + (%) PI- / PNA- (%) PI- / PNA + (%) Control 23.1 ± 3.9 ^b 42.49 + - 3.5 ^c 22.9 ± 0.5 ^c 11.4 ± 0.4 ^c AFP (0.01 mg / ml) 17.9 + 0.8 ^a 51.9 ± 1.1 ^d 21.0 ± 0.4 ^d 9.2 ± 0.6 ^d AFP (0.1 mg / ml) 20.8 ± 1.1 ^ab 46.6 ± 0.9 ^a 21.9 ± 0.5 ^a 10.6 ± 0.3 ^a AFP (1 mg / ml) 27.7 ± 0.6 ^c 33.9 ± 0.8 ^b 30.8 ± 0.5 ^b 7.5 ± 0.2 ^b p- Value p < 0.001 p < 0.001 p < 0.001 p < 0.001

As shown in FIG. 3 and Table 4, it was confirmed that the degree of acrosome damage of the thawed sperm was significantly reduced when the microorganism derived AFP protein derived from Luciferidium was added to the medium and frozen and preserved.

Experimental Example  5. Verification of effect on preservation of heart valve tissue

To evaluate the effect of preserving the heart valve tissue in the diluent prepared in Example 1, the experimental dog was euthanized, and then the normal valve tissue was isolated and stored in a preservative (diluent) for 24 hours H & E staining and TUNEL staining were performed. The results are shown in FIGS. 4 and 5, respectively.

As shown in FIGS. 4 and 5, there was no significant difference in histopathological findings between the heart valve and the heart valve stored for 24 hours in the normal heart valve and the preservative.

<110> GACHON UNIVERSITY OF MEDICINE AND SCIENCE INDUSTRY-ACADEMIC COOPERATION FOUNDATION          KOREA OCEAN RESEARCH AND DEVELOPMENT INSTITUTE <120> Cryopreservation technique using antifreeze protein from          Leucosporidium sp. <130> d <150> 11/118326 <151> 2011-11-14 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 241 <212> PRT <213> Leucosporidium sp. <400> 1 Gln Arg Asp Leu Ser Val Glu Leu Gly Val Ala Ser Asn Phe Ala Ile   1 5 10 15 Leu Ala Lys Ala Gly Ile Ser Ser Val Pro Asp Ser Ala Ile Leu Gly              20 25 30 Asp Ile Gly Val Ser Pro Ala Ala Ala Thr Tyr Ile Thr Gly Phe Gly          35 40 45 Leu Thr Gln Asp Ser Ser Thr Thr Tyr Ala Thr Ser Pro Gln Val Thr      50 55 60 Gly Leu Ile Tyr Ala Ala Asp Tyr Ser Thr Pro Thr Pro Asn Tyr Leu  65 70 75 80 Ala Ala Ala Val Ala Asn Ala Glu Thr Ala Tyr Asn Gln Ala Ala Gly                  85 90 95 Phe Val Asp Pro Asp Phe Leu Glu Leu Gly Ala Gly Glu Leu Arg Asp             100 105 110 Gln Thr Leu Val Pro Gly Leu Tyr Lys Trp Thr Ser Ser Val Ser Val         115 120 125 Pro Thr Asp Leu Thr Phe Glu Gly Asn Gly Asp Ala Thr Trp Val Phe     130 135 140 Gln Ile Ala Gly Gly Leu Ser Leu Ala Asp Gly Val Ala Phe Thr Leu 145 150 155 160 Ala Gly Gly Ala Asn Ser Thr Asn Ile Ala Phe Gln Val Gly Asp Asp                 165 170 175 Val Thr Val Gly Lys Gly Ala His Phe Glu Gly Val Leu Leu Ala Lys             180 185 190 Arg Phe Val Thr Leu Gln Thr Gly Ser Ser Leu Asn Gly Arg Val Leu         195 200 205 Ser Gln Thr Glu Val Ala Leu Gln Lys Ala Thr Val Asn Ser Pro Phe     210 215 220 Val Pro Ala Pro Glu Val Val Gln Lys Arg Ser Asn Ala Arg Gln Trp 225 230 235 240 Leu    

Claims (9)

A composition for cryopreservation of germ cells or biological tissues comprising an antifreeze protein (AFP) derived from a microorganism of the genus Lucosporium.
The cryopreservation composition of claim 1, wherein the AFP protein is composed of an amino acid sequence represented by SEQ ID NO: 1.
The composition according to claim 1, wherein the composition comprises Modena B medium as a diluent.
The method of claim 1, wherein the germ cells are selected from the group consisting of primordial germ cells, embryonic germ cells, testicular germ cells, spermatogonial stem cells and germline stem cells germline stem cells). &lt; / RTI &gt;
5. The composition for cryopreservation according to claim 4, wherein the germ cell is a sperm.
(a) collecting semen from an animal other than a human;
(b) diluting the harvested semen using a diluent;
(c) centrifuging the diluted semen to remove supernatant and obtaining a sperm deposit; And
(D) after diluting the sperm precipitate, adding and cooling the microorganism-derived antifreeze protein (AFP) of the genus Lucosporium and cooling to obtain frozen sperm; Cryopreservation method of germ cells or living tissue using the.
The method of claim 6, wherein the diluent of step (b) comprises a modified Modena B medium, which is a basic diluent, an LEY extender, which is a cold diluent, and a LEYGO diluent, which is a freeze diluent. Cryopreservation of germ cells or living tissues.
8. The method of claim 7, wherein the modified Modena B medium is comprised of glucose, EDTA, sodium citrate, sodium bicarbonate, tris, citric acid, cysteine, BSA and kanamycin sulfate. .
The method for cryopreservation of germ cells or living tissue according to claim 6, wherein the cooling in step (d) is a first cooling with liquid nitrogen vapor, followed by a second cooling with liquid nitrogen.

Images