WO2008108549A1

WO2008108549A1 - Method on long-term structural preservation of hemocyte utilizing cellular lyophilization technique

Info

Publication number: WO2008108549A1
Application number: PCT/KR2008/001119
Authority: WO
Inventors: Jootae Kim
Original assignee: Jootae Kim
Priority date: 2007-03-05
Filing date: 2008-02-27
Publication date: 2008-09-12

Abstract

This invention was made on the method of preserving cells structurally for a short or long time using lyophilization technique. The technique to preserve lyophilized cells of human for a long time has its limitation, but our research institution has found a way to preserve lyophilized cells structurally and safely for a short and long time. The model of cells to preserve is human hemocytes. Explanations will be given on the method of storing in two categories: short-term storage and long-term storage. To preserve hemocytes safely for a short time, hemocytes is incubated in 800mM trehalose buffer solution at 37°C for 15 hours. The names and concentration of cell protection material are 5 × 10-4M L-ascorbic acid, 5 × 10-5M α-lipoic acid, 5 ×× 10-5M Melatonin, 3 × 10-4M N-acetyl-L-cysteine, 10-5M δ-tocopherol, 5 × 10-5M insulin, and 10-5M prostaglandin E1. Long-term storage starts with lyophilization of incubated hemocytes, which will be added with glycerol later. Hemocytes will be incubated for 15 hours and will be added with lyophilization buffer until the hematocrit reaches to 5%. Then, the hemocytes will be cooled at a rate of 1? per minute until they get to -40?, in which temperature the hemocytes will be incubated for 30 minutes. Primary drying will be performed at -30? for 7 hours at a vacuum of 30mTorr. During secondary drying which will be performed for 6 hours, shelf temperature will be increased from -30? to 20? at a rate of 0.8? per minute using a vacuum of 50mTorr. After then, samples will be put into a specially manufactured container. The air in the container will be removed and the container will be filled with anhydrous glycerol(≥99.5%) for a long-term storage. Though a lot of studies are required to perfectly recover the function, this method ensures structural stabilization which enables semi-permanent storage. This technique to store lyophilized cells semi-permanently can be utilized in many ways in our daily lives. For example, for remembering deceased people in funeral or for commemoration between lovers. Despite various purposes in our lives, this technique has never been developed. Thus, we apply for a patent on the method and utilization of this technique.

Description

METHOD ON LONG-TERM STRUCTURAL PRESERVATION OF HEMOCYTE UTILIZING CELLULAR LYOPHILIZATION

TECHNIQUE

Technical Field

[1] The field of this invention belongs under cell preservation using lyophilization technique. If this method is utilized for preservation of eukaryotic cell, lyophilized cell can be stabilized structurally and can be preserved without cell damage over a long period of time. Background Art

[2] When the cell is dehydrated, cell loses the structural and functional water. What kind of material is necessary to protect cell from this situation. This material must substitutes water roles and it should be stabilized cell membrane. This material must substitute structural water of the cell. And this material must maintain the stability of enzymatic activity in the various oiganelle of the cell inside. This material has been selected as trehalose in this experiment. Trehalose is discovered from the microbe or the plant of part in the dehydrated environment. But eukaryotic cell will not be able to keep the cell long time with only trehalose. The following is its reason. First of all, it is difficult to load trehalose without damage of cell membrane in the eukaryotic cells, because trehalose does not go through the cell membrane in usual condition. The major challenge in using disaccharide for preservation of cells is to overcome the impermeable plasma membranes to these sugars and then to introduce the disaccharide at a sufficiently high intracellular concentration. Several experimental approaches have been developed for introducing disaccharide into mammalian cells; experiments introduced trehalose by endocytosis, experiments introduced trehalose by dimethyl sulfoxide during the transition, experiments introduce trehalose by genetically engineered pore-forming protein, experiments introduced trehalose by genetically engineered mammalian cell line, experiments intruded trehalose by high trehalose level in the extracellular space. Each methods are the merits and demerits. Method related with this study is to load high treahlose level in the extracellular space and then to increases trehalose concentration of cell inside. This method has advantage that cell membrane is not manipulated artificially. But there is a problem that cell damage increases because of high treahalose level in itself. If erythrocjtes are incubated during 24 hours, the half degree of the cells become hemolytic. This hemolytic erythrocyte should be removed one by one to use clinically, but this technique is very difficult. Moreover non-hemolytic erythrocyte can not be regarded healthy for its weakness. So using this method, technique that stabilze the cell and cell membrane should be developed.

[3] Next, the following subject is concerning problem which is happened when red blood cell be preserved during long term. Our previous study found out that damage of eu- karyotic cell in high trehalose level was similar to damage by lacking of antioxidant or by excessive oxidation. So our laboratory presumed that the reasons which were not preserved long term were following two reasons; damage of cell oiganelle by free radical and natural dischaige of biological eneigy. That is, eukaryotic cells do not have protective genetic mechanism under dehydrated state, compared to some mi- crooiganisms endurable to dehydrated circumstances. In order to preserve the cells in the receptacle for long duration, usual method is used that make the inside of the receptacle vacuum condition. Vacuum condition can do protection of microoiganisms by minimizing oxidation, but there are a problems in case that preserve eukaryotic cell; it is impossible to make high vacuum condition that gas does not exist totally, the minimum gas induce eukaryotic cell to cell oxidation because eukaryotic cell don't have genetic mechanism that stoping cellular function in the dehydrated state. This oxidation produce free radical, it destroys cell oiganelle and cell membrane. Method that solve this problem is to find the stable materials for preserving eukaryotic cell. Disclosure of Invention Technical Problem

[4] The first subject that should solve is as following. When cells are loaded with trehalose of high concentration, cell damage should be reduced to clinical usable level. To use clinically, next two conditions must be satisfied; First, The rate of cell damage must become around 5%. Second, trehalose concentration of cell inside must become more than 6OmM. It is not difficult to make cell model loaded to high trehalose level. Simply trehalose and antioxidant have only to be put on the incubated cells. But it is very difficult that determinate concentration and preparation of trehalose and antioxidant. Several kinds of antioxidant must be put on one by one and observe result one after another. Previous our study revealed that insulin increased antioxidant function on hemolysis of red blood cell. So, mixture of antioxidant and insulin were loaded on the same cell model. [5] Next second subject that should solve is as following. What kind of material can stabilize structurally eukaryotic cell and keep long term to be. This material must possess following conditions. First, the reaction of this material must be reversible. If this material has permanent binding to cell oiganelle, cell can not do cellular activity more. Second, this material has a role of freeze drying protective agent. If this material has freeze drying protective effect, it will have more desired effect than vacuum preservation. Third, this material has little water. Because freeze drying cell has about 5% water, this material must have similar water content. Fourth, this material should be low toxicity and less cell permeability. Fifth, this material must be safe. That is, this material has no explosiveness or no change by room temperature.

[6] In this study, glycerol is selected among several materials. First, glycerol is material that exist in human body. Glycerol is main component of triglyceride and phospholipid and used biological eneigy by glucose syithesis. Therefore this material has reversible reaction with human cell. Second, glycerol is freeze drying protective agent. Glycerol is used much by freeze drying protective agent next to trehalose in the natural. Third, water absorptiveness of glycerol is very high, but if there does not exits water in storage receptacle, there will be no circumstance to absorb water. And glycerol exits liquid state in room temperature. Fourth, glycerol is very low toxicity and low cell permeability. Fifth, glycerol has no explosiveness, and there is no any change of state in room temperature. Technical Solution

[7] Solution of first subject

[8] The model of cells to preserve is human erythrocytes. The antioxidants are selected all materials including with δ-tocopherol, α-lipoic acid, melatonin, N- acetyl-L-cysteine, L-ascorbic acid which is good effect from previous hemolysis experiment. Insulin is used as material which rises an effect of antioxidant. PBS (Phosphate-buffered saline, 300mOsm, pH 7.2) contained 154mM NaCl, 1.06mM KH₂ PO ₄ and 5.6mM Na₂HPO₄ ADSOL (4ffimθsm) contained 11 ImM glucose, 2mM adenine, 154mM NaCl and 4ImM mannitol. Buffer PBS(T) contained PBS and indicated amount of trehalose. Buffer ADSOL(T) contained ADSOL and K-phosphate and indicated amount of trehalose. ADSOL(T) (80OmM) contained 80OmM endotoxin- free trehalose (Hayashibara biochemical lab. Japan) and lOOmOsm ADSOL. δ- tocopherol was prepared as aqueous solution including l%(v/v) ethanol. α-lipoic acid and melatonin was prepared as aqueous solution including 0.5%(v/v) ethanol. This use of ethanol did not show no significant change in previous study of red blood cell hemolysis. Order of experiments, measuring methods, results of experiment were as followings

[9] 1. order of experiments

[10] ® Whole human blood was drawn the same day from healthy volunteer.

[11] © This whole blood was gathered to tube with EDTA(ethylenediaminetetraacetate).

[12] © Whole blood was separated to each blood corpuscle by centrifugation (329g,

14min). After spin, erythrocytes were collected from the bottom portion of the packed red cells. Then the cells were washed in PBS and centrifuged at 515g, lOmin three times. At each spin, erythrocytes were collected same method. Finally the erythrocytes were stored in PBS at 4⁰C with 30% hematocrit and used within 1 day.

[13] ® At beginning experiment, the cell washed in ADSOL(T) (800mM)and centrifuged at 515g, lOmin three times. At each spin, erythrocytes were collected same method with 30% hematocrit.

[14] © Then some of cell suspension were put to Erlenmeyer flask, time was checked and each conditions were experimented. The suspension was set as used buffer was ADSOL(T) (80OmM), temperature was 37⁰C, time was 15hours and cell protective materials were antioxidants and insulin combination in accordance with protocol. Concentrations of all materials were final concentrations of cell suspension.

[15] © All incubations contained 10 /i^penicillin-streptomycin/m^ cell suspension to vitiate any microbial growth during the overnight incubations. The working solution of penicillin- streptomycin contained 300mg penicillin G and 500 mg streptomycin in 10ml buffer.

[16] 2. methods of trehalose detection

[17] Following incubation, erythrocytes were washed three times in 900mOsm PBS

(which is isoosmotic to the loading medium) and collected by centrifugation(1960g, lmin). The loaded cells (0.5ml 30% hematocrit) were centrifugated at 350g for 3min and were mixed with 4ml of 80% methanol. The mixture was incubated at 85⁰C for 45min followed by centrifugation at 20Og for lOmin. The supernatant was collected and evaporated using a vacuum centrifuge(Labconco, USA), and dry residue was dissolved in 3mL of nano-pure water. This sample (3mL) were mixed with 6mL of anthrone reagent [2% anthrone in concentrated sulfuric acid], heated to 100⁰C for 3min, and allowed to cool. Absorbance at 620nm was measured on a spectrophotometer (Hitachi U2000, Japan)at room temperature and compared with a standard curve. Since the anthrone method detects all sugars, unloaded control erythrocytes were always treated in parallel. These values, normalized for cell count, were subtracted from the trehalose specifically and to avoid artifact due to endogenous sugars. Data are shown for at least three independent experiments.

[18] 3. Hemolysis determination

[19] Loaded erythrocytes were analyzed for RBC number, cell volume, mean corpuscular hemoglobin, and total hemoglobin using a Bechman Coulter Counter(Act Diff, USA). Percent hemolysis was determined by measuring the hemoglobin(Hb) content in the cell suspension (total Hb) and in the supernatant(free Hb) after pelleting the cells by centrifugation (196Og, lmin for isolated and loaded erythrocytes). Total hemoglobin was converted to cyanmethemoglobin using by Drabkin's reagent and the absorbance was measuring at 54Onm. The percent hemolysis was calculated using the following formula.

[20] % Hemolysis = 100 x (OD ₅₄₀ of the supernatant)/(OD₅₄₀ of total hemoglobin)

[21] 4. Statistics

[22] All chemicals were purchased from Sigma Chemical Co.(St.Louis, MO) unless otherwise noted. Data were analyzed statistically using Wilcoxon (n=5). A P value of less than 0.05 was considered significant.

[23] 5. Results

[24] Figure 1 presents the result of incubated erythrocytes in 80OmM trehalose buffer without cell protection protocol for 15 hours in 37⁰C. When erythrocytes were incubated during 15 hours (©+4. ImM), this result showed that trehalose concentration of cell interior becomed above 6OmM.

[25] Figure 2 presents degree of erythrocytes hemolysis in 80OmM trehalose buffer without cell protection protocol in 37⁰C. When erythrocytes were incubated for 15 hours(25±3.1%), erythrocytes showed 25% hemolysis. This result showed that erythrocytes were available to use clinically if only hemolytic erythrocytes were removed. Also, this result showed that 5% hemolysis happened when 3 hours passed. Therefore, cell protection protocol must decrease cell damage up to result of 3 hours hemolysis statistically.

[26] Figure 3 presents the degree of hemolysis under each conditions. A(5.2+0.69%) and

1(5.0+0.59%) showed no statistical significance, other case showed statistical significance. H(66±1.17%) case showed that hemolysis approached to 5% level. Its case had cell protection protocol consisted of δ - tocopherol, α - lipoic acid, melatonin, N- acetyl-L-cysteine, L-ascorbic acid. I case showed that hemolysis approximate nearly to 5% level. Its had cell protection protocol consisted of 10⁵M δ - tocopherol, 5 x 10⁵M α - lipoic acid, 5 x 10⁵M Melatonin, 5 x 10⁴M L-ascorbic acids, 3 x 10⁴M N- acetyl-L-cysteine, and 5 x 10 ⁵M insulins. This result showed that δ - tocopherol, α- lipoic acid, melatonin, N-acetyl-L-cysteine, L-ascorbic acid, insulin were very effective cell protection protocol.

[27] Figure 4 presents the result of incubated erythrocytes in 80OmM trehalose buffer with the final cell protection protocol (δ - tocopherol, α - lipoic acid, melatonin, N- acetyl-L-cysteine, L-ascorbic acid, insulin) in 37⁰C. It showed that concentration of cell inside beconed above 6OmM when erythrocytes were incubated for 15 hours (68+3.ImM). This value showed that there was no statistical significance to compared to value of result which was incubated for 15 hours (69+4.ImM) in Figure 1.

[28] Therefore, final setting values of conditions are requisite that names of cell protection protocol are δ - tocopherol, α - lipoic acid, melatonin, N-acetyl-L-cysteine, L-ascorbic acid, insulin, incubated time is 15hours, and incubated temperature is 37⁰C.

[29] Solution of second subject

[30] The model of cells to preserve is human hemocytes. The names and concentration of cell protection material are 5 x 10^'4M L-ascorbic acid, 5 x 10^'5M α-lipoic acid, 5 x 10^~5 M Melatonin, 3 x 10⁴M N-acetyl-L-cysteine, 10 ⁵M δ-tocopherol, and 5 x 10⁵M insulin. And the 10⁵M prostaglandin El is also used because prostaglandin El did role of stabilizing platelet in our previous experiment. PBS (Phosphate-buffered saline, 300mOsm, pH 7.2) contained 154mM NaCl, 1.06mM KH₂PO₄ and 5.6mM Na₂HPO₄ ADSOL (462mOsm) contained 11 ImM glucose, 2mM adenine, 154mM NaCl and 4ImM mannitol. Buffer PBS(T) contained PBS and indicated amount of trehalose. Buffer ADSOL(T) contained ADSOL and K-phosphate and indicated amount of trehalose. ADSOL(T) (80OmM) contained 80OmM endotoxin-free trehalose (Hayashibara biochemical lab. Japan) and lOOmOsm ADSOL. Lyophilizing buffer contained lOOmOsmol ADSOL(24mM glucose, 0.43mM adenine, 33mM NaCl, 89mM mannitol), 10OmM trehalose, 66mM K-phosphate, 15% high molecular weight hydroxyethyl starch (HES, B. Braun Medical, Irvine, CA) and 2.5% human serum albumin (HSA, Sigma- Aldrich). δ-tocopherol was prepared as aqueous solution including l%(v/v) ethanol. α-lipoic acid, melatonin and prostaglandin were prepared as aqueous solution including 0.5%(v/v) ethanol. This use of ethanol did not show no significant change in previous study of red blood cell hemolysis. All incubations contained 10 /^penicillin-streptomycin/ mi cell suspension to vitiate any microbial growth during the overnight incubations. The working solution of penicillin- streptomycin contained 300mg penicillin G and 500 mg streptomycin in 10ml buffer. Order and results of experiments are as followings. [31] 1. order of experiments

[32] ® Whole human blood was drawn the same day from healthy volunteer.

[33] © This whole blood was gathered to tube with EDTA(ethylenediaminetetraacetate).

[34] © Whole blood was separated to each blood corpuscle by centrifugation (329g,

14min). After spin, erythrocytes were collected from the bottom portion of the packed red cells, leukocytes were collected from the buffy coat, and platelets were collected from upper layer of sample. Each hemocytes were collected to same tube. Then the cells were washed in PBS and centrifuged at 515g, lOmin three times. At each spin, blood corpuscles were collected same method. Washed erythrocytes were stored in PBS at 4⁰C with 30% hematocrit and used within 1 day.

[35] ® At beginning experiment, the cell washed in ADSOL(T) (800mM)and centrifuged at 515g, lOmin three times. At each spin, blood corpuscles were collected same method and washed erythrocytes were set with 30% hematocrit.

[36] © Then some of cell suspension were put to Erlenmeyer flask. Then cell protection materials were put to flask. The suspension were set as used buffer was ADSOL(T) (80OmM), temperature was 37, time was 15 hours. Concentrations of all materials were final concentrations of cell suspension.

[37] © The conditions of lyophilizer (Labconco,USA) were set as following. Hemocytes were incubated for 15 hours and were added with lyophilization buffer until the hematocrit reached to 5%. Then, the hemocytes were cooled at a rate of I⁰C per minute until they got to -4O⁰C, in which temperature the hemocytes were incubated for 30 minutes. Primary drying were performed at -3O⁰C for 7 hours at a vacuum of 30mTorr. During secondary drying which were performed for 6 hours, shelf temperature were increased from -3O⁰C to 2O⁰C at a rate of 0.8⁰C per minute using a vacuum of SOmTorr. After then, samples were put into a specially manufactured container. The air in the container were removed and the container were filled with anhydrous glycerol(>99.5%) for a long-term storage.

[38] © The verification of result was observed by phase difference microscope(Meiji techno., Japan). Observation of slide was done under rehydrated state.

[39] 2. Results

[40] Fig 5 presents well-preserved erythrocytes generally as time passes with little damage of erythrocytes. For the 6 months, cell morphology was showed most well- preserved aspect. Also, because there is no living things that live in the anhydrous glycerol(>99.5%), if basic aseptic method is performed, the contamination to mi- crooiganism, fungus, virus will be not concerned about. However, there are some problem that totally recover function. This problem is next subject that must solve. [41] Fig 6 presents sample that be utilized for remembering deceased people in funeral.

The decoration container have lyophilized erythrocytes of the deceased. Even if all of deceased disappears with death, his blood corpuscle cells will be kept over a long period of time. After this is utilized for funeral ceremony, keeping can be with family or deceased. [42] Fig 7 presents sample that be utilized for commemoration between lovers. The decoration container is personal ornaments which is portable commemoration material of lover.

Advantageous Effects

[43] 1. One possibility is presented that eukaryotic cell can be preserved long term by lyophilized technique.

[44] 2. This experimental result is presented with one of protocol when eukaryotic cell is lyophilized.

[45] 3. It is found that combination of several cell protective materials are more useful than single specific material.

[46] 4. This result can be regarded as the data of the foundation when lyophilized eukaryotic cell will be done perfect functional revival after long term storage. Brief Description of the Drawings

[47] Figure 1 shows a graph illustraing the change of trehalose level at intervals of 3 hours when to erythrocytes were added with 80OmM trehalose in 37⁰C.

[48] Figure 2 shows a graph illustraing the degree of hemolysis at intervals of 3 hours when to erythrocytes were added with 80OmM trehalose in 37⁰C.

[49] Figure 3 shows a graph illustrating the degree of hemolysis occurred in 15 hours observation depending on each situation of cell protection protocols when to erythrocytes were added with 80OmM trehalose in 37⁰C.

[50] Figure 4 shows a graph illustrating the change of trehalose level at intervals of 3 hours observation depending on final cell protection protocols when to erythrocytes were added with 80OmM trehalose in 37⁰C.

[51] Figure 5 shows the result of long-term storage of lyophilized cells observed depending on time under the phase contrast microscope.

[52] Figure 6 shows a sample of lyophilized cells stored using this technique which can be utilized for rememberig deceased people.

[53] Figure 7 shows a sample of lyophilized cells stored using this technique which can be utilized for commemoration between lovers. [54] Keys to Abbreviations in the Figure

[55] T3: 3 hours, Tά 6 hours, T9: 9 hours, T12 12 hours, T15: 15 hours

[56] A: The condition that erythrocytes were added with 80OmM trehalose for 3 hours in

37⁰C. [57] B: The condition that erythrocytes were added with 80OmM trehalose for 15 hours in

37⁰C. [58] C: The condition that erythrocytes were added with 80OmM trehalose, 5 x 10 ⁴M L- ascorbic acid for 15 hours in 37⁰C. [59] D: The condition that erythrocytes were added with 80OmM trehalose, 5 x 10 ⁵M α- lipoic acid for 15 hours in 37⁰C. [60] E: The condition that erythrocytes were added with 80OmM trehalose, 5 x 10 ⁵M

Melatonin for 15 hours in 37⁰C. [61] F: The condition that erythrocytes were added with 80OmM trehalose, 3 x 10 ⁴M N- acetyl-L-cysteine for 15 hours in 37⁰C. [62] G: The condition that erythrocytes were added with 80OmM trehalose, 10 ⁵M δ- tocopherol for 15 hours. [63] H: The condition that erythrocytes were added with 80OmM trehalose, 5 x 10 ⁴M L- ascorbic acid, 5 x 10⁵M α-lipoic acid, 5 x 10⁵M Melatonin, 3 x 10⁴M N- acetyl-L-cysteine, 10^'5M δ-tocopherol for 15 hours in 37⁰C. [64] I: The condition that erythrocytes were added with 80OmM trehalose, 5 x 10 ⁴M L- ascorbic acid, 5 x 10⁵M α-lipoic acid, 5 x 10⁵M Melatonin, 3 x 10⁴M N- acetyl-L-cysteine, 10 ⁵M δ-tocopherol, and 5 x 10⁵M insulin for 15 hours in 37⁰C.

Best Mode for Carrying Out the Invention [65] The lyophilized cell can be preserved for remembering deceased people in funeral.

Mode for the Invention

[66] The lyophilized cell can be preserved for remembering deceased people in funeral.

[67] The lyophilized cell can be preserved for commemoration between lovers.

Industrial Applicability [68] Though this method is utilized for memory of deceased or commemoration of lover now, but in future, freezing drying method will be substitute for deep freezing method which is required high cost by advance of this method.

Claims

[1] Method of cultivating erythrocytes by compositively using L-ascorbic acid, α- lipoic acid, melatonin, N-acetyl-L-cysteine, δ-tocopherol and insulin to reduce the hemolysis of the erythrocytes, when imposing trehalose to the cells.

[2] In claim 1, method to hold the concentration of above L-ascorbic acid as 5 x I^{0 4}

M, the concentration of α-lipoic acid as 5 x 10⁵M, the concentration of melatonin as 5 x 10^'5M, the concentration of N-acetyl-L-cysteine as 3 x 10^'4M, the concentration of δ-tocopherol 10⁵M and the concentration of insulin as 5 x 10^'5M.

[3] In both of claim 1 and 2, method with a feature of cell cultivating temperature of

37⁰C.

[4] Method to utilize the lyophilized cells which were produced to preserve for a long term, in the concrete to be used as remembrance and commemoration .

[5] In claim 4, method to store human hemocytes for a long term and method with a feature of long term preservation with glycerol after lyophilizing the cells by using L-ascorbic acid, α-lipoic acid, melatonin, N-acetyl-L-cysteine, δ- tocopherol, insulin and prostaglandin El with trehalose buffer as cell protecting protocol.

[6] In claim 5, method with a feature with trehalose concentration of 80OmM, the concentration of L-ascorbic acid as 5 x 10⁴M, the concentration of α-lipoic acid as 5 x 10⁵M, the concentration of melatonin as 5 x 10⁵M, the concentration of N-acetyl-L-cysteine as 3 x 10^'4M , the concentration of δ-tocopherol as 10⁵M, the concentration of insulin as 5 x 10⁵M, the concentration of prostaglandin El as 10⁵M, and glycerol of 99.5% in an absolute state.

[7] In claim 5 and 6, method with a feature which cultivates the hemocytes at 37⁰C.