Devices, methods, and compositions useful for cryopreservation, cryopreservation, cryopreservation transport, and application of mammalian cells for treatment.
The use of a freezing medium with dimethyl sulfoxide and hyaluronic acid in a specialized container addresses viability and sterility issues in cryopreservation, enabling direct cell administration with high efficacy.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- DISCGENICS INC
- Filing Date
- 2024-09-04
- Publication Date
- 2026-06-22
Smart Images
Figure 0007877405000006 
Figure 0007877405000007 
Figure 0007877405000008
Abstract
Description
[Technical Field]
[0001] Cross-reference of related applications This application claims priority under Section 119(e) of the United States Patent Act based on U.S. Provisional Patent Application No. 62 / 466228, filed on March 2, 2017, entitled “Devices, Methods, And Compositions Useful in Cryo-Preservation Mammalian Cells,” which is incorporated in its entirety by reference.
[0002] field The processes, methods, and systems of this disclosure relate to the long-term storage of mammalian cells for medical procedures at very low temperatures without significantly reducing their viability. Such cells can be administered to patients with little or no intervention after thawing. [Background technology]
[0003] Treatment cells are either obtained directly from mammalian donors or administered to patients after selection, enrichment, amplification, etc., in cell culture. In some cases, treatment cells may be stored or transported before administration. To maintain cell viability during storage and / or transport, treatment cells can be frozen at very low temperatures to preserve and protect their therapeutic capabilities (cryopreservation or cryopreservation). This cryopreservation can enable long-term storage and / or long-duration transport with little or no impairment of therapeutic effect.
[0004] Conventional cryopreservation methods often result in inconsistent or low viability of cells after thawing. Furthermore, conventional cryopreservation methods require additional handling to transfer the cells to a device for administration and / or to remove one or more cryopreserved materials.
[0005] Subsequent manipulation of treatment cells can adversely affect their denaturation, viability, and efficacy. Furthermore, transferring treatment cells to appropriate medical devices or equipment increases the risk of contamination by undesirable bacteria, viruses, chemicals, etc. In addition, conventional freezing methods that rely on the use of animal serum can expose human recipients to the risk of contracting animal-borne diseases.
[0006] Therefore, there is a need for improved compositions and methods for use in the freezing of treatment cells, as well as means to reduce post-thawing handling. [Overview of the Initiative]
[0007] This invention discloses compositions for the cryopreservation and preservation of eukaryotic cells in therapeutic pharmaceutical compositions. The compositions of the present invention comprise a freezing medium, a cryotherapy agent (e.g., dimethyl sulfoxide), and one or more organic polymers (e.g., hyaluronic acid). The compositions of the present invention also enable the freezing of therapeutic cells with or without animal serum. The compositions and devices of this disclosure can maintain cell viability and sterility through cell freezing, long-term storage at -130°C to -196°C, thawing, and administration to a patient.
[0008] Furthermore, we disclose a container useful for freezing and thawing eukaryotic cells. In many embodiments, the container of the present invention is suitable for administering cells after thawing and requires little to no handling of the cell mixture before administration. In many embodiments, the container of the present invention comprises at least a part of a medical device such as a syringe, where the container can maintain the sterility of the cell mixture during freezing and thawing and can be used to administer the mixture to a patient who needs it. The container of the present invention may include a lumen, as well as first and second open ends, which may be configured to receive a cap, tip, plug, plunger, or other structure suitable for containing the mixture within the lumen. The container of the present invention is manufactured from a biocompatible material that maintains its integrity throughout the freezing and thawing process.
[0009] Devices, compositions, and methods for maintaining the viability of mammalian cells for treatment at low temperatures are disclosed herein, and the devices of the present disclosure are also useful for reducing cell manipulation prior to administration to a patient in need. The compositions, devices, and methods of the present disclosure maintain cell viability and thus help reduce loss of treatment efficacy.
[0010] Further objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] [Figure 1] FIG. 1 shows one embodiment of a device for freezing, storing, and applying the cell compositions of the present disclosure. The upper figure shows various parts of the unassembled device. The lower figure is a cross-sectional view of the device. [Figure 2] FIG. 2 is a diagram showing one embodiment of the assembled device of the present disclosure configured to reduce contamination during freezing, storage, transportation, and thawing of the composition of the present disclosure placed within the barrel lumen. [Figure 3] FIG. 3 compares the recovery rate and viability after storage for one month in the vapor phase of liquid nitrogen using various media with 1% hyaluronic acid between the media. The freezing container used in the experiment was a syringe system. Good recovery rates and viabilities were shown for all media used. [Figure 4] FIG. 4 shows the non-destructive measurement values of the headspace oxygen of DiscGenics syringe samples as a bar graph. It shows the values of the change in headspace oxygen concentration between the time points of T0 and T1. The headspace oxygen concentrations at both time points were processed as the average value of five repeated measurements for each syringe. The red horizontal line indicates a 3.0% decrease in oxygen, which was used as the criterion for judging the loss of container closure integrity of the syringe. [Modes for carrying out the invention]
[0012] The present invention provides compositions and methods for freezing eukaryotic cells (e.g., mammalian cells) at very low temperatures for storage and / or transport. Furthermore, it provides medical devices and apparatus that are useful for housing the frozen cells and reduce cell handling prior to subsequent administration. Furthermore, a method for freezing and storing the compositions of the present disclosure in the devices of the present disclosure is also disclosed. Herein, the freezing and storage method provides a remarkable improvement in resistance to contamination by gases, liquids and microorganisms (e.g., bacteria, fungi, viruses, etc.).
[0013] In many embodiments, the methods of the present disclosure include combining cells with a freezing medium to form a cell mixture, and then placing the cell mixture in the devices and apparatus of the present disclosure. In many embodiments, the freezing compositions of the present disclosure include a freezing medium, a cryotherapy agent, and an organic polymer. In many embodiments, the cells are placed in a container prior to freezing, where the container is part of a medical device useful for administering the cells. In such embodiments, the container is configured to withstand very low temperatures and thawing without substantially affecting the viability and sterility of the cells or the function of the device.
[0014] The present disclosure provides compositions, devices, and methods for freezing and thawing an appropriate amount of cells for use in treatment after thawing. The cells for treatment of the present disclosure have a viability of 50% to 100% after thawing (i.e., 60 to 100% of the cells have the ability to grow and divide), are sterile, and can be administered immediately. In many embodiments, about 50 to 100% of the cells are recovered from the container and administered to a patient. In many embodiments, the methods of the present disclosure help to substantially reduce the loss rate of closure integrity to less than 20%, less than 10%, less than 5%, or less. Surprisingly, the applicant has found that a polymer syringe plunger alone is not sufficient to protect the sterility of the compositions of the present disclosure. This is because at very low temperatures such as those disclosed for the freezing and storage of the cells of the present disclosure, the plunger allows gas exchange, thereby allowing contamination by biological substances such as viruses, fungi, bacteria, and other microorganisms.
[0015] In many embodiments, the compositions, methods, and devices of the present disclosure can be analyzed by measuring the expression of one or more indicator genes or proteins, such as the production of one or more cytokines, cell cycle markers, or extracellular matrix components, which helps to maintain the efficacy of the cells for treatment of the present disclosure.
[0016] The present disclosure also provides for freezing cells at high density (e.g., about 1.0×10 3 cells / ml to 5.0×10 8 cells / ml) with or without animal serum while maintaining the viability, identity, and dispersibility of the cells in the composition. In some embodiments, the cell density is about 100×10 3 , 0.5×10 6 , 1×10 6 , 1.5×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 10×10 6 , 20×10 6 , 30×106 , 40×10 6 , 50×10 6 , 100×10 6 , 200×10 6 , 300×10 6 , 400×10 6 , 500×10 6 , 600×10 6 , or 700×10 6 Larger than that, approximately 750 x 10 6 , 700×10 6 , 600×10 6 , 500×10 6 , 400×10 6 , 300×10 6 , 200×10 6 , 100×10 6 , 50×10 6 , 40×10 6 , 30×10 6 , 20×10 6 , 10×10 6 , 6×10 6 , 5×10 6 , 4×10 6 , 3 x 10 6 , 2×10 6 , 1.5×10 6 , 1 x 10 6 , 0.5 × 10 6 , or 200×10 3 It can be smaller than that.
[0017] In most cases, the cells used for treatment are 0.5 × 10⁶ 6 cells / ml~1.0×10 7 It can be frozen at a cell density of cells / ml. In some embodiments, the density of cells for treatment in the cell mixture is approximately 1.0 × 10⁶. 6 ~9.0×10 6 Cells / ml, for example, approximately 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 × 10 6Larger than cells / ml, approximately 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, and 1.1 × 10⁻¹⁰ 6 It can be smaller than cells / ml.
[0018] Cells can be frozen in single or multiple dose volumes or quantities. In some embodiments, doses range from approximately 0.1 ml to 5.0 ml, for example, approximately 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1.0 ml, 1.1 ml, 1.2 ml, 1.3 ml, 1.4 ml, 1.5 ml, 1.6 ml, 1.7 ml, 1.8 ml, 1.9 ml, 2.0 ml, 2.1 ml, 2.2 ml, 2.3 ml, 2.4 ml, 2.5 ml, 2.6 ml, 2.7 ml, 2.8 ml, 2.9 ml, 3.0 ml, 3.1 ml, 3.2 ml, 3.3 ml, 3.4 ml, 3.5 ml, 3.6 ml, 3.7 ml, 3.8 ml, 3.9 ml, 4.0 ml, or 4. The volume may be greater than 5 ml / dose and approximately 5.0 ml, 4.5 ml, 4.0 ml, 3.9 ml, 3.8 ml, 3.7 ml, 3.6 ml, 3.5 ml, 3.4 ml, 3.3 ml, 3.2 ml, 3.1 ml, 3.0 ml, 2.9 ml, 2.8 ml, 2.7 ml, 2.6 ml, 2.5 ml, 2.4 ml, 2.3 ml, 2.2 ml, 2.1 ml, 2.0 ml, 1.9 ml, 1.8 ml, 1.7 ml, 1.6 ml, 1.5 ml, 1.4 ml, 1.3 ml, 1.2 ml, 1.1 ml, 1.0 ml, 0.9 ml, 0.8 ml, 0.7 ml, 0.6 ml, 0.5 ml, 0.4 ml, 0.3 ml, or less than 0.2 ml / dose. In some embodiments, one, two, three or more doses may be frozen in a single device.
[0019] cell The cells used in the compositions, devices, and methods of this disclosure include mammalian cells, treatment cells, such as treatment human cells. The cells of this disclosure can be obtained from or administered to a variety of tissues. In many embodiments, treatment cells can be obtained from intervertebral disc tissue and administered to the intervertebral disc. In many embodiments, various types of cells, such as myofibrillators, muscle cells, chondrocytes, epithelial cells, osteocytes, osteoclasts, progenitor cells, stem cells, etc., can be obtained from and / or administered to a variety of tissues, including muscle, liver, heart, lung, pancreas, articular cartilage, tendons, ligaments, intervertebral discs, bone, thymus, thyroid gland, or lymph nodes. In some embodiments, the cells of this disclosure can be derived from articular cartilage, cardiac tissue, or bone.
[0020] The compositions, devices, and methods of this disclosure are useful for treating a variety of diseases and conditions, including but not limited to degenerative disc diseases, disc injuries, burns, lacerations, cardiac and muscular injuries, and bone fractures / separations.
[0021] composition In some embodiments, treatment cells are collected from cell culture medium prior to freezing. In one embodiment, cells are collected and pelletized by centrifugation. The pelleted cells may then be resuspended in the freezing composition of the Disclosure to form a cell mixture. In many embodiments, the freezing composition of the Disclosure may comprise a freezing medium, a cryotherapy agent, and an organic polymer.
[0022] Culture medium Freezing media (or cryoprotective media) may contain one or more additives, including but not limited to animal serum (e.g., fetal bovine serum (FCS or FBS)) and cryoprotective agents (i.e., highly water-soluble and low-toxicity substances). Cryoprotective agents added to freezing media can promote cell survival by mitigating or preventing cell damage during the freezing and thawing process.
[0023] Various commercially available freezing media can be used to prepare the compositions of the present invention for use in the methods of the present disclosure. For example, in some embodiments, animal serum may not be used, and in such embodiments, "xeno-free" media, such as Profreeze, FreesIS, Cryostor, etc., can be used instead of animal serum-containing media. In various embodiments, the freezing media of the present disclosure may also contain one or more further components, such as proteins, such as albumin. In one embodiment, human, mammalian, or synthetic serum albumin (HSA) may be included in the freezing medium. In many embodiments, the amount of freezing medium may correspond to the remainder fraction, which does not contain the cryotherapy agent and organic polymer. In some embodiments, the amount of frozen medium is approximately 60% to approximately 98.9%, for example, more than approximately 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and less than approximately 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 8%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, or 70%. In one embodiment, the amount of frozen medium is approximately 91.5%.
[0024] The culture medium of this disclosure may also contain further components such as: amino acids (e.g., glutamine, arginine, or asparagine), vitamins (including, but not limited to, one or more B vitamins, e.g., vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, or vitamin B12), transition metals (including, but not limited to, nickel, iron (e.g., ferric or ferrous), or zinc), and other culture medium components. The culture medium of the present invention may also be supplemented as needed with: hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources. In some aspects, the frozen culture medium of the present invention contains proteins derived from plants or animals. In some embodiments, the freezing medium of the present invention does not contain plant or animal-derived proteins. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art.
[0025] Freezing agent Cryoprotectants (or cryoprotective agents) are chemical substances that help cells maintain viability during the freezing and thawing processes. Cryoprotectants generally enter cells to promote a decrease in intracellular osmotic pressure and protect intracellular proteins from denaturation. One example of a cryoprotectant that can cross the cell membrane is DMSO (dimethyl sulfoxide). Because DMSO can cross the cell membrane, it can also be released from cells after thawing. Other cryoprotectants include glycerol, alginates, and polyvinyl alcohol.
[0026] The concentration of the cryotherapy agent used can range from approximately 1% (by volume or weight) to approximately 20%. In many embodiments, the concentration of the cryotherapy agent can be approximately 7.5%, for example, in the case of DMSO. In many embodiments, the concentration of DMSO used can be lowered to minimize the amount of DMSO administered to the patient being treated with the treatment cells.
[0027] polymer The freezing composition contains an organic polymer. The organic polymer of this disclosure is useful for protecting cells during freezing and for functioning as a matrix for the treatment cells when administered to a patient after thawing. In many embodiments, the organic polymer is a glycosaminoglycan, such as hyaluronic acid, sodium hyaluronate, or hyaluronan. In many embodiments, the concentration of the organic polymer is about 0.1% to 10% by weight of the mixture. In a preferred embodiment, the concentration of the organic polymer is about 1.0%.
[0028] method The cell mixtures of this disclosure can be placed in a suitable container designed to withstand very low temperatures (i.e., below about -100°C). In some embodiments, the frozen composition may be cooled before being combined with the cells for treatment. In many embodiments, the cells may be frozen at -20°C to -80°C for 5 to 120 minutes before being subjected to long-term storage. In some embodiments, for long-term storage, the composition and device may be stored at about -80°C or about -196°C. For example, the method of the Disclosure may include freezing cells in the Device of the Disclosure at approximately -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -75°C or -80°C for a period of time longer than approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes or 180 minutes, and shorter than approximately 240 minutes, 180 minutes, 120 minutes, 105 minutes, 90 minutes, 75 minutes, 60 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes or 15 minutes. In many cases, the temperature of the composition can be lowered at a rate greater than approximately 0.1°C / min, 0.2°C / min, 0.3°C / min, 0.4°C / min, 0.5°C / min, 1°C / min, 2°C / min, 3°C / min, 4°C / min, 5°C / min, 6°C / min, 7°C / min, 8°C / min, 9°C / min, or 10°C / min, but less than approximately 15°C / min, 10°C / min, 9°C / min, 8°C / min, 7°C / min, 6°C / min, 5°C / min, 4°C / min, 3°C / min, 2°C / min, 1°C / min, 0.5°C / min, 0.4°C / min, 0.3°C / min, 0.2°C / min, or 0.1°C / min.
[0029] The methods disclosed herein may involve one-step, multi-step, or ramped freezing. In many cases, after freezing cells, the temperature ranges from approximately -135°C to approximately -196°C, for example, approximately -130°C, -135°C, -140°C, -145°C, -150°C, -155°C, -160°C, -165°C, -170°C, -171°C, -172°C, -173°C, -174°C, -175°C, -176°C, -177°C, -178°C, -179°C, -180°C, -181°C, -182°C, -183°C, -184°C, -185°C, -186°C, -187°C, -188°C, -189°C, -190°C, -191°C, -192°C, -193°C, -194°C, -195°C, or above -196°C. It can be stored in the vapor phase of liquid nitrogen, where the temperature can be as low as approximately 196°C, -197°C, -196°C, -195°C, -194°C, -193°C, -192°C, -191°C, -190°C, -189°C, -188°C, -187°C, -186°C, -185°C, -184°C, -183°C, -182°C, -181°C, -180°C, -179°C, -178°C, -177°C, -176°C, -175°C, -174°C, -173°C, -172°C, -171°C, -170°C, -165°C, -155°C, -150°C, -145°C, -140°C, and higher than -135°C.
[0030] The cell compositions of this disclosure can be stored for long periods of time. In some embodiments, cells can be stored for longer than about 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years or longer, and for shorter periods than about 5 years, 4.5 years, 4 years, 3.5 years, 3 years, 2.5 years, 2 years, 1.5 years, 1 year, 52 weeks, 48 weeks, 44 weeks, 40 weeks, 36 weeks, 32 weeks, 28 weeks, 24 weeks, 20 weeks, 16 weeks, 12 weeks, 10 weeks, 8 weeks, or 6 weeks. In many embodiments, the survival rate of stored cells after thawing is higher than approximately 90%, and lower than approximately 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 80%, and 75%, and higher than approximately 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
[0031] Cells, compositions, and devices can be transported at a variety of temperatures. In some embodiments, frozen cells can be transported in a variety of media containing liquid nitrogen, vapor-phase nitrogen, etc. In some other embodiments, the methods of the present disclosure may include long-term storage and transport at temperatures from -80°C to -196°C.
[0032] Prior to freezing, the cells are resuspended in the freezing composition and placed in a container, under conditions that maintain the sterility and composition of the mixture and prevent the formation of voids or spaces between the mixture and the container. In some embodiments, a portion of the composition may be pre-filled in the device of the present invention, and the cells may be added to the composition in the container. In many embodiments, the container may be at least part of a medical device, such as a syringe barrel, but may be any container suitable for maintaining cell viability at low temperatures. In many embodiments, the container may be useful for directly administering or applying treatment cells to a patient. In some embodiments, the container may be implantable and / or biodegradable. In some embodiments, as will be detailed later, the medical device of the present invention may be a container, such as a syringe barrel, that can be capped or sealed to prevent gas exchange between the composition and nitrogen vapor or other gases surrounding the device.
[0033] The methods of this disclosure may be useful for increasing the rate at which cell temperature is reduced. Increasing the rate of temperature reduction may help maintain cell viability by avoiding the damaging side effects of freezing (e.g., dehydration). According to this disclosure, the thawing rate can also be increased compared to conventional methods. In many embodiments, thawing may be performed by placing the container at room temperature or ambient temperature (e.g., about 20°C to about 25°C) and allowing the cell mixture to become liquid. In some other embodiments, thawing may be performed on ice or using a heating device. In some embodiments, the cell mixture can be brought to a liquid state, i.e., to a temperature above approximately 0°C, in a time shorter than approximately 2 hours, 110 minutes, 100 minutes, 95 minutes, 90 minutes, 85 minutes, 80 minutes, 75 minutes, 70 minutes, 65 minutes, 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, or 20 minutes, and longer than approximately 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, or 115 minutes. In some embodiments, the cell mixture is administered to the patient at a temperature lower than approximately 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, or 20°C, and higher than approximately 15°C, 20°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or 36°C, before the cell mixture reaches body temperature (i.e., approximately 37°C).
[0034] Medical devices Cells can be placed in a medical device or container prior to freezing. In some embodiments, the medical device may be a syringe barrel or lumen, and the composition may be sealed within the barrel by one or more seals, plungers, caps, or other suitable barriers against gas exchange and / or contamination by material or organisms around the syringe. Freezing, storing, and dispensing the cells of the Disclosure in a syringe device may help reduce the costs associated with the production, transport, and application of the cells of the Disclosure. For example, using vials may be more time-consuming and costly because the cells must be transferred to a syringe for application. The use of syringes also eliminates the need for the cells of the Disclosure to pass through two or more needles, thus reducing exposure to forces such as shear forces that may affect potency. Furthermore, if the cells of the Disclosure are transferred from a storage container to an applicator, there may be a loss of the sample remaining in the original container. Figure 1 shows one embodiment of the container device of the Invention, which is a syringe.
[0035] In Figure 1, the disclosed device 100 may include a syringe barrel 200, a plunger rod 300, a plunger 400, and a needle end cap 500. An optional barrel cap 600 is also shown. In many embodiments, the syringe may not include a plunger rod. The barrel may have a barrel flange 215 and a barrel orifice 220 defining an opening to the barrel lumen 225, a first plunger end 210, and a second needle end 230. The barrel orifice has a diameter d1, and the lumen has a diameter D LThe barrel flange end may define a substantially flat surface surrounding a barrel orifice having a diameter larger than the outer diameter of the barrel. A needle hub 240 may be located at or near the needle end, which may define a needle hub lumen 241 defined by an inner surface 242 including a plurality of raised ridges 243 for receiving a cap or lure. A tip 245 may be located at the center of the needle hub lumen to receive a needle (not shown) or optionally a needle end cap 500. The tip defines a lumen 250 having a first inlet orifice 251 that is in fluid communication with the barrel lumen, and a second outlet orifice 252 that may help transfer the composition in the barrel lumen to the needle (not shown). The second outlet orifice has a diameter d2 which may be selected to minimize the shear force as the cell composition passes through the tip lumen to the syringe needle (not shown). In various embodiments, the tip lumen may have a constant diameter equal to d2, or it may have a varying diameter, with the first inlet orifice defining a diameter smaller than d1 but larger than d2. The tip lumen has an inner diameter D of the barrel lumen, which can be approximately 1.0 to 10.0 mm. LIt has a smaller inner diameter than that. In many embodiments, the barrel lumen has an inner diameter of approximately 6.0, 6.1, 6.2, 6.3, or 6.4 mm, and the tip lumen may have an inner diameter greater than approximately 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, or 2.4 mm, and smaller than approximately 2.5 mm, 2.4 mm, 2.3 mm, 2.2 mm, 2.1 mm, 2.0 mm, 1.9 mm, 1.8 mm, 1.7 mm, 1.6 mm, 1.5 mm, 1.4 mm, 1.3 mm, 1.2 mm, 1.1 mm, 0.9 mm, 0.8 mm, 0.7 mm, or 0.6 mm. The tip may also specify a length L1 that is smaller or larger than, for example, about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, or 2.4 mm, and is larger or smaller than the length L2 of the needle hub lumen. Thus, the tip may protrude from the needle hub lumen (if its length is longer than the needle hub lumen) or be recessed (if its length is shorter than the needle hub lumen). Some embodiments of the syringe of disclosure include a needle end cap or needle, while some embodiments do not. The needle end cap 500 may be configured to securely seal the tip and prevent gas exchange, leakage of cellular composition from the barrel lumen, and / or entry of gas, microorganisms, or other foreign matter into the barrel. In the embodiment shown in Figure 1, the needle end cap may have an outer surface 510 that defines a plurality of connector ridges 515 that fit into complementary structures on the inner surface of the needle hub lumen. In the embodiment shown in Figure 1, the needle end cap further defines an internal tip lumen 520 that receives the tip on the barrel.
[0036] A plunger rod can be connected to a plunger in various ways. For example, in one embodiment shown in Figure 1, the plunger rod may include a plunger connector 310 at one end and a plunger head 320 at the other end. The plunger connector may be configured to be fixedly connected to the plunger. In the embodiment shown in Figure 1, the plunger connector defines a screw 315 having a plurality of screw tines 315. The plunger may define a plunger receiving structure 415 for fixed connection to the plunger connector on the plunger rod. The embodiment shown in Figure 1 has a plunger receiving configured as a lumen for receiving the plunger connector screw of the plunger rod. The plunger may have an outer surface 420 configured to contact the lumen of the barrel. In the embodiment of Figure 1, the plunger includes a plurality of ridges 425 on its outer surface.
[0037] The applicant surprisingly found that polymers fail to maintain an airtight seal within the syringe barrel when the syringe is stored at temperatures below approximately -80°C. In many cases, this failure of polymer plungers is not affected by the removal of the plunger rod from the plunger; that is, incomplete sealing is also observed in applications where the plunger is fixedly connected to the plunger rod. The applicant also surprisingly showed that the use of a polymer cap at or near the flange orifice of the barrel can help prevent gas exchange and / or contamination. As shown in Example 2 below, gas exchange / leakage is not prevented by placing an additional plunger in the barrel. Rather, sealing the flange end of the barrel can significantly reduce the failure rate. Other methods for sealing the flange end of the syringe include placing other suitable polymer and non-polymer seals at or near the flange end of the barrel. In one embodiment, an adhesive can be placed between the polymer cap and the flat surface of the flange to facilitate sealing, and in several other embodiments, the cap can be sealed to the flat surface using other methods known to those skilled in the art.
[0038] Figure 2 shows one embodiment of the device of the present disclosure configured to maintain closure integrity and reduce contamination of the composition by liquids, gases, microorganisms, or other substances. In this embodiment, the barrel orifice is sealed at least partially at the barrel end orifice by the arrangement of a barrel cap 600. In some embodiments, the cap may be held in place by one or more structures or methods to prevent gas exchange and / or the entry of contaminants into the barrel lumen. The barrel cap may be manufactured from any compound or element that can result in a seal to the barrel flange and / or barrel orifice to prevent contamination of the barrel lumen. In some embodiments, additional structures, such as ridges (e.g., provided on the needle end cap), polymer adhesives, metal sealants, etc., may be used to assist in the fixation of the barrel cap.
[0039] The plungers, needle end caps, and barrel caps of this disclosure may be manufactured from a variety of polymeric and nonpolymeric materials suitable for use. In some embodiments, the structures may be manufactured from silicone, polypropylene, butyl rubber, natural rubber, or other suitable materials. In many embodiments, the barrel lumen, plungers, needle end caps, and / or barrel caps may further include the application of coatings, layers, or substances, which may serve to seal and reduce friction. In some embodiments, for example, the coating may be silicone, PTFE (polytetrafluoroethylene), or other suitable materials well known in the art for medical applications.
[0040] The medical devices of this disclosure are useful for freezing, storing, thawing, and administering treatment cells. In many embodiments, the devices of this disclosure include a body having a first open end and a second open end, and a lumen located between the open ends and connecting them in fluid communication. The first open end may define a sealing cap or an applicator, such as a needle, configured to be attached and removed detachably. The tip may define an interior and exterior that are in fluid communication with the lumen. The exterior of the tip may include one or more structures designed to help secure the needle, such as a Luer lock. In some other embodiments, the cap and needle may be press-fitted.
[0041] The second opening may define an opening configured to receive the treatment cells of the present disclosure in a frozen composition so that they can be positioned within a lumen. The second opening may further be configured to receive a cap, plunger, plug, or other structure designed to fit within the wall of the second opening and movably seal. The wall of the second opening may continue with the wall of the lumen. The cap, plunger, plug, or other structure may be designed to be connected (in some embodiments detachably connected) to a rod portion that can help apply force to the cap, plunger, plug, or other structure, thereby allowing the cap, plunger, plug, or other structure to move relative to the wall of the lumen.
[0042] The devices of this disclosure are designed to maintain their functionality after being stored in contact with one or more components of a frozen composition at very low temperatures. For example, the caps, plungers, plugs, or other structures of this disclosure, together with a second open end and / or lumen wall, can substantially maintain fluid sealing during freezing, storage, and thawing to help prevent contamination of treatment cells placed in the lumen.
[0043] The second open end and the lumen wall are designed to allow the smooth movement of a cap, plunger, plug, or other structure toward them while substantially maintaining fluid sealing. In some embodiments, the lumen wall may include a coating that can prevent chemical interactions between the device and the freezing composition. In some other embodiments, the composition of the lumen wall may be sufficient to prevent chemical interactions. Chemical interactions may refer to reactions that can result in changes to either the device composition or the freezing composition, which can reduce or alter the treatment effect of the cells and / or the freezing composition.
[0044] The devices of this disclosure may be manufactured from a variety of materials. In some embodiments, the materials used to manufacture the devices of this disclosure are resistant to excessive expansion, contraction, breakage, cracking, shattering, etc., during freezing and thawing. In one embodiment, the devices of this disclosure may include glass substitute polymers, such as cyclic olefin polymers (e.g., crystal zenith), or cycloolefin polymers and copolymers. In many embodiments, the devices of this disclosure may include one or more transparent materials so that the treatment cells and cryopreserved compositions can be monitored.
[0045] The lumen wall is thin enough to allow controlled and rapid freezing and thawing. In many embodiments, the material of the device's lumen may be capable of effectively conducting heat to allow rapid freezing and thawing of cells for treatment.
[0046] Caps, plugs, plungers, or other materials may be made of polymer compounds that resist expansion, contraction, cracking, shattering, etc., during freezing and thawing. In some embodiments, the polymer compound comprises one or more organic polymers, such as isoprene or latex. In some embodiments, lubricants or coatings may be applied to the caps, plugs, plungers, or other materials to facilitate the maintenance of a seal against the lumen wall.
[0047] definition "Culture medium" and "cell culture medium" refer to solutions for maintaining the viability of eukaryotic cells in culture and in situ (for example, after administration to a patient requiring such cells). The culture mediums of this disclosure include nutrients utilized for cell growth (e.g., one or more such as serum, serum substitutes, glucose, galactose, etc.) and one or more further components (e.g., hormones, peptides, vitamins, essential amino acids, non-essential amino acids, minerals, salts, buffers, etc.).
[0048] "Freezing medium" may refer to a medium containing one or more additional components useful for maintaining the viability of eukaryotic cells during and after freezing, such as cryoprotective agents.
[0049] Further aspects of the present invention are described below: [Section 1] Freezing medium; Freezing agents; and Organic polymers A pharmaceutical composition for maintaining the viability of treatment mammalian cells, including the above. [Section 2] The pharmaceutical composition according to item 1 above, wherein the freezing agent comprises dimethyl sulfoxide. [Section 3] The pharmaceutical composition according to item 1 or 2 above, wherein the frozen culture medium does not contain animal serum. [Section 4] A pharmaceutical composition according to any one of items 1 to 3 above, wherein the polymer is hyaluronic acid. [Section 5] A pharmaceutical composition according to any one of items 1 to 4 above, wherein the cells are derived from intervertebral disc tissue, skin, muscle, intestine, bone marrow, nerve, liver, heart, lung, pancreas, articular cartilage, bone, thymus, thyroid gland, or lymphoid tissue. [Section 6] A pharmaceutical composition according to any one of items 1 to 5 above, wherein the cells are derived from intervertebral disc tissue, articular cartilage, cardiac tissue, or bone. [Section 7] Devices for freezing and thawing treatment cell mixtures, including the following: It is a container, A first open end having a first diameter, A second open end having a second diameter smaller than the first diameter, A lumen including a lumen wall having a lumen diameter similar to the first diameter and in fluid communication with the first and second open ends. A container that includes, and in which the container is made of one or more biocompatible materials that maintain structural integrity when frozen at or below approximately -130°C and when thawed; A first polymer seal, which is fitted into the lumen and configured to provide sealing contact with the lumen wall; A first polymer cap designed to seal the first open end; and A second polymer cap designed to seal the second open end. [Section 8] The container according to item 7, wherein the container defines a syringe barrel and the first polymer seal is a piston or plunger. [Section 9] The container according to item 7 or 8, wherein the container is made of a transparent polymer. [Section 10] The container according to any one of items 7 to 9 above, wherein the container is made of a cyclic olefin polymer. [Section 11] The container according to any one of the above paragraphs 7 to 10, wherein the second open end specifies a Luer lock that can receive a syringe needle of a suitable structure when the second polymer cap is removed. [Section 12] Methods for freezing mammalian cells for treatment, including the following: Combining multiple treatment cells and compositions to form a mixture; The mixture is placed in a container for a medical device having a first opening and a second opening; The first polymer seal is placed in contact with the mixture; To form an airtight seal by placing a first polymer cap in the first opening and a second polymer cap in the second opening; To reduce the temperature of the mixture to -80°C or below; Maintain the mixture at a temperature below approximately -80°C for at least 30 days; thereafter, at least approximately 50% of the cells will have grown in cell culture and undergone at least one division. [Section 13] The method according to item 12, wherein the mixture is maintained in the vapor phase of liquid nitrogen at a temperature below about -130°C or for at least part of the 30 days. [Section 14] The method according to item 12 or 13, wherein at least about 90% of the cells have grown and divided at least once after 30 days. [Section 15] The method according to any one of items 12 to 14 above, wherein after maintaining the cells, the mixture is thawed to a liquid form at a temperature of about 20 to 40°C. [Section 16] The method according to any one of items 12 to 15 above, wherein the treatment cells are derived from human intervertebral disc tissue. [Section 17] The aforementioned container A first open end having a first diameter, A second open end having a second diameter, A lumen including a lumen wall that is in fluid communication with the first and second open ends. The method according to any one of items 12 to 16 above, wherein the container is made of one or more biocompatible materials that are frozen at less than about -130°C and maintain structural integrity when thawed. [Section 18] The method according to any one of items 12 to 17, wherein the device prevents at least the exchange of nitrogen gas when the device is stored at a temperature below -80°C. [Section 19] The method according to any one of items 12 to 18 above, wherein the cells are derived from intervertebral disc tissue, skin, muscle, intestine, bone marrow, nerve, liver, heart, lung, pancreas, articular cartilage, bone, thymus, thyroid gland, or lymphoid tissue. [Section 20] The pharmaceutical composition according to any one of items 12 to 19 above, wherein the cells are derived from intervertebral disc tissue, articular cartilage, cardiac tissue, or bone. While several embodiments have been disclosed, further embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the present invention can be modified in various obvious aspects without departing from its essence and scope. Therefore, the detailed description should be interpreted as illustrative and not limiting.
[0050] Any references disclosed herein, whether patent or non-patent, are incorporated herein by reference as if they were included in their entirety at the cited location. In the event of any inconsistency between the references and the specification, including definitions, the specification shall prevail.
[0051] While this disclosure is described with a certain degree of specificity, it is understood that this disclosure is provided as an example and that changes may be made in detail or structure without deviating from the essence of this disclosure as defined in the claims. [Examples]
[0052] Recovery and viability in frozen compositions The recovery and viability of cells in various culture media were compared. Cells were frozen in the container device disclosed above and stored in the gas phase of liquid nitrogen for one month. The container used in this experiment was a syringe system. By storing the cell mixture in the syringe system disclosed above, the cell mixture can be directly administered to the subject with minimal handling after thawing.
[0053] As shown in the bar graph in Figure 3, good recovery rates and viability were achieved with all of the tested frozen compositions. [Examples]
[0054] Integrity of closure Surprisingly, initial tests revealed that freezing and thawing samples in syringes at very low temperatures, such as the temperature of liquid nitrogen vapor, resulted in oxygen loss in the samples. This indicated that the polymer plunger and / or tip cap were insufficient to maintain the seal during freezing, storage, and thawing. This malfunction was observed both when the plunger rod was in place and when it was removed.
[0055] To investigate the cause of this malfunction, a series of configurations were tested using various syringes and closures. The objective of this test was to evaluate container closure integrity (CCI) by non-destructive analysis of the oxygen concentration [%] and total pressure [torr] in the syringe headspace of multiple syringe configurations stored at very low temperatures. For this test, several syringes representing eight different syringe configurations were prepared. These syringes were filled with samples and stored in a very cold storage chamber for approximately 4.5 days. Under these conditions, if container closure integrity is compromised, a decrease in oxygen concentration in the syringe headspace may occur. This decrease in oxygen concentration is thought to be a result of gas leakage and exchange between the nitrogen-rich storage environment and the composition inside the syringe.
[0056] As a control, one syringe for each configuration was stored at room temperature in a nitrogen-purged, airtight chamber. This negative control sample was designed to investigate the effect of very low storage temperatures on gas exchange / leakage.
[0057] Headspace oxygen concentration was measured using tunable diode laser absorption spectroscopy (TDLAS).
[0058] Sample set To test the closure integrity of different configurations, various syringes, plungers, and stoppers were combined (Figure 4 and Table 1). Seven replicate samples were prepared for each sample format listed in Table 1 below.
[0059] For each replica, the oxygen concentration and total pressure in the headspace of each syringe were measured before and after storage.
[0060] [Table 1-1] [Table 1-2] [Table 1-3]
[0061] Methods and materials Sample preparation and manipulation In this study, PBS was used as the test composition. A Gilson P1000 Pipetman was used to fill each syringe with PBS (Gibco; Cat#: 20012-027; pH 7.2). Care was taken to avoid forming droplets on the inner surface of the syringe barrel. A Dabrico vented placement tool (Dabrico; Cat#: MS-25) was used to insert the specified plunger into the syringe.
[0062] In some sample formats, additional barriers were created by inserting 081 and 420 stoppers. Specifically, these stoppers were manually inserted into the syringes of sample formats 2 and 7, respectively.
[0063] Sample numbers 1-5 represented the test group for each sample format. Sample number 6 represented a positive control with a predetermined defect. The defect in this sample number 6 replica was created by inserting the syringe needle through the syringe plunger (and stopper, if present). Specifically, a 23-gauge × 1-inch needle was used for all sample formats except sample format 7, and an 18-gauge × 1.5-inch needle was used for sample format 7 (necessary to penetrate both the plunger and stopper). Sample number 7 for each sample format represented the negative control stored at room temperature in a nitrogenous environment, as described above.
[0064] Initial values (T0) for both headspace oxygen concentration and total pressure were measured on the day the sample formats were prepared. On the second day, samples 1-6 of each sample format were placed in a -20°C freezer for 3 hours, and then transferred to a -80°C freezer. After placing samples 1-6 at -80°C for approximately 4 hours, they were stored in a Cryoport Express® High Volume Dry Vapor Shipper (all excess liquid nitrogen was removed). The temperature of the dry vapor shipper was monitored using an Apollo IV digital thermometer (UEi; Cat#: DT304) and maintained at a constant -177°C (96K) for approximately 4.5 days of frozen storage. Sample 7 was stored in an airtight container purged with pure nitrogen and kept at ambient room temperature (approximately 23°C) for approximately 4.5 days.
[0065] After storage for approximately 4.5 days, samples 1-6 were removed from frozen storage. The samples were immediately placed in a separate nitrogen-purged airtight chamber and thawed and equilibrated to room temperature by being left at ambient room temperature (approximately 23°C) for about 2 hours.
[0066] Both headspace oxygen concentration and total pressure were measured at time T1 in all syringes of all formats. Five measurements were taken for each sample. Figure 4 shows a graph illustrating the change in headspace oxygen concentration from T0 to T1.
[0067] Measurement of headspace oxygen A frequency-modulated, tunable semiconductor laser absorption spectrometer (Lighthouse) was used to measure headspace oxygen in each replica. This method, known as TDLAS, provides non-invasive and rapid gas analysis of the headspace within a sealed container, giving both the number density of a specific gas and the total headspace pressure.
[0068] Prior to sample analysis, six oxygen standards with known oxygen concentrations were measured five times each consecutively to validate the instrument's performance. Based on the absolute value of the measurement error (the difference between the known value and the measured value) and the measurement precision (the standard deviation of multiple measurements for each standard), the uncertainty of headspace oxygen concentration measurements for this test was established as ±0.8% atm.
[0069] Measurement of headspace pressure Headspace pressure was measured using an FMS-Moisture / Pressure Headspace Analyzer (Lighthouse) according to standard measurement procedures. Briefly, the instrument was turned on and warmed up for at least 30 minutes under a nitrogen purge of at least 1 standard L / min before starting the measurement session. Calibration was performed using known pressure / humidity standards.
[0070] Prior to sample analysis, to validate the instrument's performance, five consecutive measurements were taken of pressure standards with known total pressures (10 standards for one format and 8 standards for another). Based on the absolute value of the measurement error (the difference between the known value and the measured value) and the measurement precision (the standard deviation of multiple measurements for each standard), the uncertainty of the headspace total pressure measurement for this test was established as the greater of ±6 torr and ±7% of the measured value.
[0071] result The results of non-destructive headspace oxygen measurements are summarized in Figure 4. The change in headspace oxygen concentration between time points T0 and T1 is shown for each syringe in nine different sample formats. The red horizontal line indicates a 3.0% oxygen decrease, which was used as the criterion for determining incomplete syringe closure (see the discussion section below). The uncertainty of headspace oxygen concentration measurements for the standards used in this study was established as ±0.8% atm, based on the performance of the oxygen standards. The results of each headspace parameter measured for each syringe sample are summarized in Table 3. The uncertainty of headspace total pressure measurements for each sample format was established as the greater of approximately 6 torr from the measured value or approximately 7% from the measured value, based on the performance of the Lighthouse pressure standards.
[0072] [Table 3] TIFF0007877405000004.tif179159 [Table 3 (continued)] TIFF0007877405000005.tif121162
[0073] Discussion and Conclusion Headspace oxygen concentration and total pressure were successfully measured for all syringes in each of the nine configurations (Table 1). The uncertainty of the headspace oxygen concentration measurement in this study was established as ±0.8% atm based on the oxygen standard. The uncertainty of the corresponding total pressure measurement was established as the greater of ±6 torr and ±7% of the measured value, based on the performance of the pressure standard. Since the syringes contained liquid water, the headspace was saturated with water vapor, thus giving the maximum FMS signal obtainable in the described syringe configurations.
[0074] The measurement results are summarized in Table 3. The changes in headspace oxygen concentration [%] and total pressure [torr] after low-temperature storage are also shown in the same table. Under the above conditions, if the container seal integrity is broken in a particular syringe, the decrease in oxygen concentration in its headspace can occur through three possible processes. The first process causing the decrease is that when the temperature drops, nitrogen vapor surrounding the syringe in the very cold storage chamber may leak into the syringe through the damaged (defective) part, thereby diluting the total oxygen in the headspace and thus decreasing the oxygen concentration. The second is that after the syringe reaches thermal equilibrium at a storage temperature of approximately 177°C, diffusion may occur, allowing oxygen molecules to leak from the syringe headspace into the nitrogen environment of the very cold storage chamber. The third is that when the syringe is removed from very cold storage, the temperature rise in the leaking syringe may create excessive pressure in the headspace, and this excess pressure may tend to push out the syringe headspace gas containing the remaining oxygen. It should be noted that if the container seal integrity is breached and resealed during a temperature rise, excessive headspace pressure may be maintained. The “Plunger Malfunction” column in Table 3 shows cases where the presence of excessive pressure in the syringe headspace pushes the syringe plunger back.
[0075] Based on the oxygen level changes measured in the negative control (sample number 7), a decrease in headspace oxygen concentration exceeding 3.0% was used as the criterion for container closure failure. Furthermore, syringes in which the plunger was pushed back due to a rise in syringe temperature ("plunger malfunction") were also considered to have container closure failure. Finally, a headspace pressure increase exceeding 100 torr was defined as an additional criterion for determining that CCI loss had occurred.
[0076] The results for individual syringes, shown in Figure 4 and Table 3, indicate that 29 out of 45 syringe samples stored at low temperatures lost their closure. As expected, all eight positive controls (sample number 6) also failed to meet the criteria.
[0077] For sample formats 1, 4, 5, and 8, all five replicas lost CCI. For sample format 3, four out of five test syringes lost CCI. Each of these formats used only a plunger to seal the syringe barrel. Interestingly, sample format 8 included two plungers. Sample format 7, which included a stopper at the barrel end, was the most effective in preventing CCI loss.
Claims
1. Methods for freezing mammalian cells for treatment, including the following: Combining multiple treatment cells and compositions to form a mixture; The mixture is placed in a container for a medical device having a first opening and a second opening; Placing the first polymer seal in contact with the mixture; To form an airtight seal by placing a first polymer cap in the first opening and a second polymer cap in the second opening; To reduce the temperature of the mixture to -80°C or below; Maintain the mixture at a temperature of -80°C or lower for at least 30 days; thereafter, at least about 50% of the cells have grown in cell culture and undergone at least one division.
2. The method according to claim 1, wherein the mixture is maintained in a liquid nitrogen vapor phase at -130°C or below for at least a portion of the 30 days.
3. The method according to claim 1 or 2, wherein at least about 90% of the cells have grown and divided at least once after 30 days.
4. The method according to any one of claims 1 to 3, wherein after maintaining the cells, the mixture is thawed to a liquid form at a temperature of about 20 to 40°C.
5. The method according to any one of claims 1 to 4, wherein the treatment cells are derived from human intervertebral disc tissue.
6. The aforementioned container A first open end having a first diameter, A second open end having a second diameter, A lumen including a lumen wall that is in fluid communication with the first and second open ends. The method according to any one of claims 1 to 5, wherein the container is made of one or more biocompatible materials that maintain structural integrity when frozen at -130°C or below and when thawed.
7. The method according to any one of claims 1 to 6, wherein the device prevents at least the exchange of nitrogen gas when the device is stored at a temperature below -80°C.
8. The method according to any one of claims 1 to 7, wherein the cells are derived from intervertebral disc tissue, skin, muscle, intestine, bone marrow, nerve, liver, heart, lung, pancreas, articular cartilage, bone, thymus, thyroid gland, or lymphoid tissue.
9. The method according to any one of claims 1 to 8, wherein the cells are derived from intervertebral disc tissue, articular cartilage, cardiac tissue, or bone.