Microinjection of cryoprotectants for preservation of cells

Details Microinjection of cryoprotectants for preservation of cells Microinjection of cryoprotectants for preservation of cells

2001-05-16

2004-01-06

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of storing cells in a viable state

C435S001300, C435S002000, C435S325000, C436S018000

Reexamination Certificate

active

06673607

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the preservation of biological tissue using microinjection of intracellular protective agents containing sugar to preserve cells by freezing and/or drying.
BACKGROUND OF THE INVENTION
In recent years, chemotherapy and radiation therapy of patients with cancer has been increasingly successful and sustained remissions have been achieved. However, the chronic side effects of these therapies to the reproductive systems of long-term survivors is of particular concern. These effects for women include depletion of ovarian germ cells and sterility. Due to the potential loss of future fertility of those exposed to cancer therapy, a need for oocyte banking has developed. Oocyte freezing, when combined with in vitro fertilization, may be beneficial to women desiring future fertility who are anticipating loss of gonadal function from extirpative therapy, radiation, or chemotherapy. Oocyte freezing may also provide a possible alternative to human embryo freezing, thus avoiding many of the legal and ethical problems encountered in embryo freezing.
The first successful cryopreservation of human embryos was achieved in 1983 and embryo freezing is now a routine procedure. In contrast, very limited success has been reported with cryopreservation of human oocytes. Only five successful pregnancies have been reported with more than 1500 cryopreserved oocytes. Therefore, the current methods of freezing are still considered experimental and novel approaches are needed to overcome the difficulty encountered by cryopreservation of the human oocyte.
Traditional cryopreservation techniques include penetrating cryoprotectants at concentrations of 1 to 2M with, for example, dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol, followed by a slow freezing rate (0.3 to 0.5° C./min). Typically, oocytes are damaged due to long-term exposure to deleterious freezing conditions, including excessive dehydration and high electrolyte concentrations. An alternative approach, called vitrification (i.e. formation of glassy material without crystallization of ice, uses high concentrations of cryoprotectant mixtures (6 to 8M) followed by rapid cooling in order to avoid the lethal effects of freezing on oocytes.
Though an attractive alternative, vitrification procedures suffer from the toxic and osmotic effects of high cryoprotectant concentration on sensitive cells. Neither of these two approaches (slow freeze-thaw and rapid vitrification) has resulted in a reliably successful outcome for cryopreservation of human oocytes. Thus, there is a need for a reliable technique for human oocyte storage. In order to provide the preservation of mammalian cells necessary for application of living cells as a therapeutic tool in clinical medical care, new protocols for preserving living nucleated cells using low levels of non-toxic preservation agents and having simple procedures applicable to a variety of cells must be developed.
SUMMARY OF THE INVENTION
The purpose of the present invention is to allow the storage of living cells in a dormant state and the subsequent recovery of the cells to an active state. This method involves microinjecting into the cytoplasm of a cell a protective agent that is substantially non-permeating with respect to mammalian cell membranes and that maintains the viability of the cell such that it can be stored in a temporarily dormant state and substantially restored to an active state. The microinjected cell is subjected to conditions that cause it to enter a dormant state and is stored in this dormant state. The stored cell can be subsequently restored to an active state. This method has the advantage of allowing any mammalian cell to be stored until it is needed under conditions that cause minimal, if any, adverse side-effects in the cell.
Thus, the invention, in some embodiments, provides a method for preserving living cells that begins with microinjecting a protective agent containing an effective sugar into the cell, preferably an oocyte. Other preferred cells that may be preserved include differentiated cells, such as epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-cells, T-cells, erythrocytes, macrophages, monocytes, fibroblasts, or muscle cells; and undifferentiated cells, such as embryonic, mesenchymal, or adult stem cells. In one preferred embodiment, the differentiated cells remain differentiated after they are recovered from a frozen or dried state, and the undifferentiated cells remain undifferentiated after they are recovered. The cells can be haploid (DNA content of n; where “n” is the number of chromosomes found in the normal haploid chromosomes set of a mammal of a particular genus or species), diploid (2n), or tetraploid (4n). Other cells include those from the bladder, brain, esophagus, fallopian tube, heart, intestines, gallbladder, kidney, liver, lung, ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, or uterus. The cells may be from a human or non-human mammal, such as a monkey, ape, cow, sheep, big-horn sheep, goat, buffalo, antelope, oxen, horse, donkey, mule, deer, elk, caribou, water buffalo, camel, llama, alpaca, rabbit, pig, mouse, rat, guinea pig, hamster, dog, or cat.
The method of the invention may advantageously use low levels, less than or equal to about 6, 5, 4, 3, 2, or 1 M, or even less than about 0.4 M of preservation agent, and may use a sugar alone as the preservation agent, or sugar in combination with a conventional cryoprotectant, or in combination with other intracellular sugars or extracellular sugars. More preferably, the cytoplasmic concentration of the sugars is less than 0.3, 0.2, 0.1, 0.05, or 0.01 M after microinjection and before freezing or drying of the cell. The extracellular concentration of the sugars is preferably less than 0.3, 0.2, 0.1, 0.05, or 0.01 M after dilution into a liquid medium containing the cell. If the cell is grown on a solid support, such as an agar plate, the concentration of the sugars in the preservation agent that is contacted with the cell is preferably less than 0.3, 0.2, 0.1, 0.05, or 0.01 M. In other preferred embodiments, the final concentration of extracellular sugar in the medium containing the cell is at least 2, 3, 4, 5, or 10 fold greater than the cytoplasmic concentration of intracellular sugar after microinjection and before freezing or drying of the cell. The intracellular and extracellular preservation agents may be the same or different molecular species.
Preferred protective agents include sugars such as monosaccharides, disaccharides, and other oligosaccharides. Preferably, the agent is substantially non-permeable such that at least 50, 60, 70, 80, 90, or 95% of the agent does not migrate across the plasma membrane into or out of the cell, by active or passive diffusion. Preferred sugars have a glass transition temperature of the maximally freeze-concentrated solution (Tg′) that is at least −60, −50, −40, −30, −20, −10, or 0° C. Examples of such sugars are those listed in FIG.
8
. The Tg′ of other sugars may be routinely determined using standard methods such as those described by Levine and Slade (J. Chem. Soc., Faraday Trans. 1, 84:2619-2633, 1988). The sugar or conventional cryoprotectant with a Tg′ below −50° C. can be combined with a sugar with a Tg′ above −50° C. such that the resulting mixture has a Tg′ of at least −60, −50, −40, −30, −20, −10, or 0° C., and this mixture is used for cryopreservation.
Suitable monosaccarides include those that have an aldehyde group (i.e., aldoses) or a keto group (i.e., ketoses). Monosaccharides may be linear or cyclic, and they may exist in a variety of conformations. Other sugars include those that have been modified (e.g., wherein one or more of the hydroxyl groups are replaced with halogen, alkoxy moieties, aliphatic groups, or are functionalized as ethers, esters, amines

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