1. Field of the Invention
The present invention relates to freeze-dried blood cells, stem cells, and platelets, and a manufacturing method for the same which does not require a special cooling medium and/or device during cooling such that only a small amount of energy is required wherein mixing/removal of a cryoprotectant such as glycerol or the like is unnecessary.
2. Relevant Art
With regard to maintaining one's own blood for transfusion in times of necessity, and rare blood stored in case of emergency for persons possessing rare blood types, blood for general transfusion, as well as the blood components of the aforementioned all require storage. This storage period usually involves time periods of at least one month.
In the long-term storage of blood and blood components, a freezing process for freezing the aforementioned to -80.degree. C. using an electric freezer, a freezing process using liquid nitrogen (approximately -196.degree. C. storage), and a freezing process using liquid helium (-270.degree. C. storage) represent known methods. All of these blood storage methods require a special cooling medium, container for cooling, and a supply of energy for executing the aforementioned cooling. In addition, before freeze storage, it is necessary to mix some cryoprotectants, compounds that protect living cells against the freezing damage of growing ice crystals or some stresses, such as some glycerol solutions with the blood, and then remove this cryoprotectant at the time of usage after thawing.
In addition, in the long-term storage of blood and blood components, these long-term storage methods of blood and blood components are conducted in the following manner.
An example in which concentrated red blood cells (hematocrit value=55.about.90%) are frozen will be explained hereinafter (hematocrit value is the proportion of blood cell component to overall blood component: the normal hematocrit value of blood of a healthy person is approximately 45.about.50%).
1) Using sodium citrate or heparin as an anticoagulant, blood drawn from donors is centrifuged and separated in order to remove plasma and the buffer coat thereby producing concentrated red blood cells possessing a hematocrit value of 55.about.90%.
2) An equivalent amount of cryoprotectants comprising mainly glycerol is then added to these concentrated red blood cells. This storage solution, for example, may comprise the following:
______________________________________ a) glycerol 60 g b) 70% sodium lactate 2.57 g c) KCl 0.02 g d) NaCl 0.26 g ______________________________________
The aforementioned composition is dissolved in purified water, and brought to a final volume of 100 ml.
3) The blood after mixing is then placed in a storage vessel and frozen. In the freezing of blood, placed in a container used normally in transfusion (volume 200 ml), the average cooling speed depends on the process; however, a fast process will cool at approximately 100.degree. C./min., while a slow process will cool at several .degree.C./min.
A correlation exists between the cooling speed of the blood, glycerol concentration, and hemolysis (corresponding to the death rate of blood cells) of the blood following thawing. When attempting to restrict the hemolysis to less than a fixed value, in the case of a rapid cooling speed, it is enough to use glycerol of a comparatively low concentration, while in the case of a low cooling speed, it is necessary to use glycerol of a comparatively high concentration. In practice, the following two methods are employed.
a) High-concentration glycerol low freezing speed method PA1 b) Low-concentration glycerol high freezing speed method PA1 pre-treating a liquid selected from the group consisting of blood including blood cells, bone marrow fluid (hemopoietic stem cells), and platelets in blood plasma, with a solution containing at least one substance selected from the group consisting of saccharide, biopolymer, acid and acid salt; PA1 conducting granulation of said pre-treated liquid into a granules of a first predetermined size; PA1 performing rapid cooling of said granules of the first predetermined size into a frozen product of a second predetermined size; and PA1 drying said frozen product by means of sublimation of a water content therein.
In a), the cryoprotective solution containing glycerol of the composition mentioned in 2) above is added to an equivalent amount of blood, poured into an appropriate container, and placed in an electric freezer at -80.degree. C. At this time, the blood inside the aforementioned container reaches a temperature of -80.degree. C. in a time period ranging from ten minutes to two hours, hence the average cooling speed is 1.degree..about.10.degree. C./min. For example, when cooling from room temperature (20.degree. C.) for the one unit of the concentrated red blood cells (.apprxeq.200 ml),
______________________________________ average cooling speed = {20 - (-80)}/10.about.{20 - (-80)}/120 = 10.about.0.83 .apprxeq. 1.about.10.degree. C./min. ______________________________________
In the case of b), for example, when a low-concentration glycerol solution of 28% of glycerol, 3% of mannitol, and 0.65% of NaCl is added to an equivalent amount of blood, poured into an appropriate container, and dipped in liquid nitrogen, according to this method, the blood inside the aforementioned container reaches the temperature of the liquid nitrogen (-196.degree. C.) in 1.about.2 minutes from room temperature. Therefore, the average cooling speed is 2.degree..about.4.degree. C./sec for one unit of the blood (.apprxeq.200 ml).
______________________________________ average cooling speed = {20 - (-196)}/60.about.{20 - (-196)}/120 = 3.6.about.1.8 .apprxeq. 2.about.4.degree. C./sec. ______________________________________
4) At the time of usage, after removing the blood from a freezer, rapid thawing is conducted using a water bath of approximately 40.degree. C.
5) After thawing, the glycerol is removed by washing the blood using a physiologically isotonic saline solution such as brine or the like. This process requires several hours to perform.
With regard to the method for granulating the blood, conventionally, a method for performing granulation by means of performing drop-wise addition using a gas in liquid nitrogen is known.
For example, details of the aforementioned method may be found by referencing T. Sato (A Study of Red Blood Cell Freeze Storage Using Droplet Freezing [translated]; Journal of the Hokkaido University School of Medicine, vol. 58, No. 2, pages 144.about.153 (1983)). In this method, a process in which the inflow strength of air is adjusted to produce droplets of various sizes is employed as the method for granulation. In the above case, the size of the droplets for mixing with a gas are rather large, e.g., 0.7.about.2.8 mm as shown in FIG. 4 of the aforementioned document. The recovery rate [100--(the hemolysis of red blood cells)] is also poor, varying in the 35.about.70% range. In addition, in the same Figure, when the size is reduced to 0.5 mm, the recovery rate fails below 20% which is impractical for use.
In this method, a double tube formed from a polyethylene inner tube possessing an inner diameter of 0.4 mm surrounded by an outer tube possessing an inner diameter of 3 mm is employed. In this manner, blood is introduced from the inner tube, while gas is introduced from the outer tube. Using Bernoulli's theorem, blood is introduced by generating negative pressure at the output end of the inner tube, and gas is mixed therein. This mixture is then added dropwise to liquid nitrogen which is positioned underneath the aforementioned. The granular size of the blood drops is determined according to the diameter of the inner tube, while the dropwise speed is determined by means of the flow amount of gas flowing into the outer tube.
Consequently, according to this method, as shown in the aforementioned FIG. 4, only large granules of 0.7 mm to 2.8 mm, or granules of an extremely limited range can be formed. As shown in the same Figure, when the diameter of the inner tube is enlarged, the granular size increases, which, in turn, leads to an increase in the hemolysis. In addition, reducing the diameter of the inner tube causes blinding, and dropwise addition of the blood then becomes impossible.
In other words, in the conventional granulation method, due to the use of a simple system of a negative pressure based on Bernoulli's theorem, sufficient control over the granulation cannot be obtained. Hence, disadvantages exist such as a large granular size ranging from 0.7 mm.about.2.8 mm, such that the formation of granules of 0.5 mm or less is not possible. In the case of a granular size of 0.5 mm, control is difficult leading to an extremely poor recovery rate (i.e., hemolysis is high).
The conventional methods for storing blood described above possess the following drawbacks.
1) The cooling process requires a special cooling medium and container, which in turn require the supply of a large amount of energy. For example, liquid nitrogen and liquid helium are expensive, costing 100 yen per liter and 3,000 yen per liter, respectively, and also require a special container with regard to sealing and cooled insulation.
2) A cryoprotective solution consisting mainly of glycerol is employed, and thus it is necessary to set aside time for removing the glycerol after thawing and before use.
As described in the aforementioned, the conventional blood storage methods pose problems in that these methods require a special cooling medium and container, as well as a large supply of energy. Furthermore, removal of the glycerol is complex and requires time; hence, these methods cannot be applied in case of emergencies or urgent situations.
In addition, the conventional blood freezing method employing a droplet process possesses disadvantages in that, in addition to the large granular size (0.7 mm to 2.8 mm) obtained, a high hemolysis results which cannot be applied to practical use.
USPTO Backgrounds
USPTO Backgrounds