WO2014188365A1

WO2014188365A1 - A portable device for maintaining viability of biological material and a method thereof

Info

Publication number: WO2014188365A1
Application number: PCT/IB2014/061613
Authority: WO
Inventors: Palanivel VASANTHI; Kulkarni NAVEEN
Original assignee: Polyclone Bioservices Pvt. Ltd.
Priority date: 2013-05-22
Filing date: 2014-05-22
Publication date: 2014-11-27

Abstract

The present disclosure relates to portable device for maintaining viability of biological material. The portable device comprising receptacle configured into an inner compartment and an outer compartment such that a gap (22) is created between the inner compartment and the outer compartment. The inner compartment contains culture media and the biological material. A plurality of heat packs is placed in the gap to maintain temperature of the culture media. The reaction chamber is sealed to the inner compartment. The reaction chamber comprises an acid-base mixture and solvent for generating carbon dioxide by a chemical reaction inside the reaction chamber. A filter is provided in the receptacle to enable controlled supply of carbon dioxide generated from the reaction chamber to the inner compartment. The carbon dioxide supplied to the inner compartment maintains pH value of the culture media and maintains the viability of the biological material for predetermined hours during transportation.

Description

A PORTABLE DEVICE FOR MAINTAINING VIABILITY OF

BIOLOGICAL MATERIAL AND A METHOD THEREOF"

TECHNICAL FIELD

The present disclosure generally relates to medical device. In particular, the disclosure relates to a portable device for maintaining viability of a biological material.

BACKGROUND AND PRIOR ART OF THE DISCLOSURE

Regenerative medicine is the process of creating living, functional tissues to repair or replace tissue or organ function lost due to damage, or congenital defects. Cell therapies associated with regenerative medicine currently being investigated include those using mature cells and stem cells from the actual patient (autologous cells), those involving cells from other humans (allogenic cells), and even transgenic cells from other species (xenogeneic cells). As the first step, a biopsy will be performed at the hospital. Post the biopsy, surgeon will send the biopsy collected from patient's body to processing Centre, where the cells from biopsy are isolated, differentiated and cultured for few weeks. Finally, the cultured cells which have the regenerative capability are transported back to the hospital and will be implanted on the damaged or defect site of the patient. Thus, now this implant will start to form cells at the damaged area and repair the defect.

The success of regenerative medicine approach depends upon the availability of practicable methods and means to maintain the viability of the cellular material (biopsy or expanded cells) for prolonged durations between the isolation and expansion stages and the expansion and implantation stages. For a successful transplantation and shorter post-therapy hospitalization it is important to maintain at least 80% of the viable cells when the biopsy reaches the processing centre. Existing methods do not provide the necessary conditions that maintain the viability of cells for longer duration that is required for transportation from hospitals to processing centers and cultured cells from processing centers to hospitals. This has necessitated that hospitals which offer these services need to have centralized facilities where stem cell isolation, processing and transplantation are all handled at one location. Patients are required to travel to these hospitals and get admitted for therapy. This makes the treatment expensive and logistically inaccessible to a majority of patients across the country.

These constraints can be overcome if a practicable device can be designed that maintains the viability of the cellular material (biopsy or expanded cells) for prolonged durations between the isolation and expansion stages and the expansion and implantation stages. If this is achieved it would no longer be required to have centralized processing and transplantation facilities. Local clinics across the country can act as cell isolation/ collection centers which would collect the cells from the patients and ship it to the larger hospitals that have the tissue engineering facility, via the portable device. Once received, the hospitals would expand the cells and ship it back to local clinics for transplantation back to the patient. Since the cells would remain viable for a longer period, this would facilitate their transportation back and forth, eliminating the need for patients to travel and get hospitalized for long durations. Such a device would help increase the reach of this therapy to a vast unserved patient pool, across the entire country.

Over the years, researchers have tried to address this need by different methods (cryopreservation & freeze drying) and transportation devices (Rainovet al., 2000; Zhang et al., 2009). These methods fulfill partial conditions for transport, compromising on the viability of cells. The existing devices are either labor-intensive, require technical expertise for handling the biological material or are not suitable for transportation.

Since most cell therapy products (CTP) and tissue engineered products are infused or processed shortly after collection, and in some cases this may be delayed for up to 48 hours, the biopsy transportation device of the instant disclosure is designed in such a way that it mimics standard growth conditions so that the viability, sternness of stem cells and integrity of biopsy and bio-cellular material, as well as cultured cells are maintained throughout the transportation period for maximum up to 96-120 hours.

SUMMARY OF THE DISCLOSURE The shortcomings of the prior art are overcome and additional advantages are provided through the present disclosure. Additional features and advantages are realized through the techniques of the present disclosure. Other embodiments and aspects of the disclosure are described in detail herein and are considered a part of the claimed disclosure.

In one embodiment of the present disclosure, a portable device for maintaining viability of a biological material is provided. The device comprising a receptacle configured into an inner compartment and an outer compartment concentrically arranged into one another such that a gap is created between the inner compartment and the outer compartment. The inner compartment contains a culture media and the biological material inside a culture media. A plurality of heat packs placed in the gap created between the inner compartment and the outer compartment to maintain temperature of the culture media present in the inner compartment. A reaction chamber is configured in the portable device at a predetermined location. The reaction chamber comprises an acid-base mixture and a predetermined solvent for generating carbon dioxide by a chemical reaction inside the reaction chamber. At least one filter is configured in the receptacle to enable controlled supply of the carbon dioxide generated from the reaction chamber to the inner compartment; and at least one lid to close the inner compartment and the outer compartment, wherein the inner compartment is hermitically closed.

In one embodiment, the reaction chamber is connected to top surface of the inner compartment or bottom surface of the inner compartment or sides of the inner compartment (11a) or inside the inner compartment (11a).

In one embodiment, the heat packs are provided in between base of the outer compartment and base of the inner compartment.

In one embodiment, the reaction chamber is configured to the at least one lid closing the inner compartment. In one embodiment, the lid is configured with a plurality of ports for providing the acid-base mixture and the solvent to the reaction chamber.

In one embodiment, the acid-base mixture is in form selected from a group comprising a premixed powder form or gel form or capsule forms.

In one embodiment, the receptacle is made of biocompatible material selected from a group comprising medical grade plastics, selected from a group comprising Polycarbonate, Polyetherimide, Polysulfone, PolyEtherEther-Ketone, Polyphenylene sulfide, Polyphenylsulfone, Polyacetal copolymer, Polyester, Polypropylene, Polytetrafluoroethylene, Poly(methyl methacrylate) (PMMA), Acrylonitrile butadiene styrene (ABS), Silicone.

In one embodiment, the portable device is configured to maintain the viability of the biological material selected from a group comprising a cellular material, a tissue material and its combination thereof.

In one embodiment, the heat packs maintain temperature of the culture media at about 37°C for a time of about 96-120 hours but not limited to for maintaining viability of the biological material.

In one embodiment, the plurality of filters is provided in junction of the reaction chamber and the inner compartment or in the junction of the inner compartment and the outer compartment.

In one embodiment of the present disclosure, a method for maintaining viability of a biological material using the portable device is provided. The method comprising acts of providing an acid-base mixture and the predetermined solvent inside the reaction chamber at predetermined molar concentration for generating carbon dioxide; and supplying the generated carbon dioxide into the inner compartment in a controlled manner using the plurality of filters for maintaining pH of the culture media, thereby to maintain viability of the biological material for predetermined hours during transportation from one place to another place. In one embodiment, the acid-base mixture is an acid-carbonate mixture and the predetermined solvent is water.

In one embodiment, the carbon dioxide supplied to the inner compartment is in concentration ranging from 1% v/v to 10% v/v.

The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

The novel features and characteristic of the disclosure are set forth in the appended claims. The invention itself, however, as well as a preferred mode of use, further objectives and advantages thereof, will best be understood by reference to the following detailed description of an illustrative embodiment when read in conjunction with the accompanying figures. One or more embodiments are now described, by way of example only, with reference to the accompanying drawings wherein like reference numerals represent like elements and in which:

Fig. 1 shows a portable device for maintaining viability of a biological material according to one embodiment of the present disclosure.

Fig. 2shows a portable device for maintaining viability of the biological material according to an exemplary embodiment of the present disclosure.

Fig. 3 shows a portable device for maintaining viability of the biological material according to another exemplary embodiment of the present disclosure.

Fig. 4shows a portable device for maintaining viability of the biological material according to another exemplary embodiment of the present disclosure. Fig. 5 shows graphical representation of cell viability studies conducted on cells stored at different conditions for different duration and cells that are transported using the portable device according to an embodiment of the present disclosure.

Fig. 6shows qualitative and quantitative analysis of the pH of the culture media and stem cell marker analysis for the cells that are transported using the portable device according to an embodiment of the present disclosure.

The figures depict embodiments of the disclosure for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.

DETAILED DESCRIPTION OF THE DISCLOSURE

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this disclosure.

Referral Numerals

Viability of biological materials during transport from one place such as a hospital to another place such as a processing center and vice versa depends upon the aspects such as cell storage medium, temperature, pH and CO₂ generation. This is further influenced by shipping conditions and duration of the transport. The viability of biological material can be retained for longer durations during transportation by supplying 3-5% of CO_2. The CO₂ can be generated using an acid-base system along with the biological material immersed in a culture media to supply basic nutrients for sustenance of the cells or biological materials at ambient temperature and also to ensure pH stability of the culture media. The portable device for maintaining viability is designed in such a way that it maintains standard growth conditions (typically, an ambient temperature with 5% CO₂ for mammalian cells) throughout the transportation period which would keep the cells in its native state.

Environmental parameters essential for all mammalian cell cultures are temperature of aboutl8°C -37°C, Humidity >95% (to prevent evaporation of the nutrient media supplied for the growth of the cells where the cell culture flasks are not sealed completely), pH of about 7.2to 7.4 of the culture media is maintained by maintaining a constant level or quantity of CO₂. For most cell cultures, the culture media is formulated to maintain a pH of 7.4 in 5%CO₂. Fig. 1illustrates a portable device (10) for maintaining viability of a biological material according to an exemplary embodiment of the present disclosure. The portable device (10) comprising a receptacle (11) configured into an inner compartment (11a) and an outer compartment (11b). The inner compartment (11a) and the outer compartment (l ib) are concentrically arranged into one another such that a gap (22) is created between the inner compartment (11a) and the outer compartment (11b). In one embodiment, the receptacle (11) is made of biocompatible material selected from a group comprising, medical grade plastics, selected from a group comprising Polycarbonate, Polyetherimide, Polysulfone, PolyEtherEther- Ketone, Polyphenylene sulfide, Polyphenylsulfone, Polyacetal copolymer, Polyester, Polypropylene, Polytetrafluoroethylene, Poly(methyl methacrylate) (PMMA), Acrylonitrile butadiene styrene (ABS), Silicone. The inner compartment (11a) contains a culture media (17) and the biological material (18) inside the culture media (17). The portable device (10) also comprises a plurality of heat packs (12) placed in the gap (22) created between the inner compartment (11a) and the outer compartment (l ib) to maintain temperature of the culture media (17) present in the inner compartment (11a). In one embodiment, the Heat packs (12) provided in the outer compartment (11b) contacts sides and a base of the inner compartment (11a). The Heat packs (12) are also provided in between base of the outer compartment (11b) and base of the inner compartment (11a). The Heat packs (12) maintain the temperature of the inner compartment (11a) which in turn maintains the temperature of the culture media at about 37°C. The Heat packs provided at the bottom of the inner compartment (11a) ensures that the temperature of the inner compartment is maintained for long hours such as about 72 hours. A reaction chamber (14) is selectively and hermitically sealed to the inner compartment (11a) at predetermined location in the receptacle (10). The reaction chamber (14) comprises an acid -base mixture (19) and a predetermined solvent (20) for generating carbon dioxide by a chemical reaction inside the reaction chamber (14). In one embodiment, the reaction chamber (14) is connected to a top surface of the inner compartment (11a) or a bottom surface of the inner compartment (11b). The fig. 1 shows the reaction chamber (14) is configured to a lid (16). The lid (16) is used to cover the inner compartment (11a) and the outer compartment (11b). In one embodiment different lids can be used to close inner compartment (11a) and the outer compartment (11b). The figure shows the reaction chamber (14) is partially inside the inner compartment. The configuration of the reaction chamber (14) at the top surface of the inner compartment (11a) facilitates the equal distribution of the CO₂ generated by the acid -bicarbonate system is stable for 72 hours. Also, as the CO₂is generated at top, the CO₂ tends to come down as it is heavier than air. Thus theCO₂gets distributed to the inner compartment (11a) equally. Once the CO₂is in touch with the media (liquid), it will get dissolved with the culture media very effectively. So there is no exclusive gas distribution system is required. Also, the CO₂ is fully oxidized and therefore is extremely stable by nature. The portable device (10) also comprises at least one filter (15) is provided at predetermined place in the receptacle (11) to enable controlled supply of the carbon dioxide (21) generated from the reaction chamber (14) to the inner compartment (11a). In one embodiment, the filters (15) contains but not limited to hydrophobic membrane. During CO2 generation one of the by products from the chemical reaction is water, so hydrophobic membranes in the filter prevents the water from entering to the culture media from the reaction chamber.

In one embodiment, at least one filter (15) is provided in junction of the reaction chamber (14) and the inner compartment (11a) or in the junction of the inner compartment (11a) and the outer compartment (11b). The carbon dioxide (21) generated in the reaction chamber (14) is supplied to the inner compartment (11a) to maintain pH value of the culture media (17) and thereby maintains the viability of the biological material (18) for predetermined hours during transportation from one place to another place. In one embodiment, the biological material (18) selected from a group comprising but not limited to a cellular material and tissue material as mentioned in the Table2and its combination thereof. In one embodiment, the portable device (10) further comprises at least one lid (16) which is configured with a plurality of ports (16a) for providing the acid-base mixture

(19) and the solvent (20) inside the reaction chamber (14).

In one embodiment, the heat packs (12) maintain temperature of the culture media (17) at about 37°C for time of about 72 hours for maintaining viability of the biological material (18).

In one embodiment, the filters that can be used in the device is 0.2 μm pore sized membranes (Biocompatible) and syringe filters are one of the types of such filters. The number of filters and number ports for the filters are dependent on the size of the device and the internal pressure. In one embodiment, the number of filters and ports would be in the range from 1-10.

In one embodiment of the present disclosure, a method of maintaining viability of the biological material (18) using the portable device (10) is provided. Firstly, the method comprising acts of providing an acid-base mixture (19) and the predetermined solvent

(20) inside the reaction chamber (14) at predetermined molar concentration for generating carbon dioxide (21), and secondly supplying the generated carbon dioxide

(21) into the inner compartment (11a) in a controlled manner using the plurality of filters (15) and maintaining the temperature of the culture media using heat packs (12) at about 37°C for maintaining pH of the culture media (17). Thereby viability of the biological material (18) is maintained for predetermined hours during transportation from one place to another place.

In one embodiment, the acid-base mixture (19) is an acid-carbonate mixture and the predetermined solvent (20) is water. The carbon dioxide (21) supplied to the inner compartment (11a) is in concentration ranging from 1% v/v to 10% v/v.

In one embodiment of the present disclosure, the solvent (20) used is sterile double distilled water. The chemical reaction occurring within the portable device (10) is:

C₆H₈O₇+3NaHCO₃→Na₃ (C₆H₅O₇) + 3H₂O+3CO₂ In one embodiment, the acid-base mixture added into the reaction chamber is in predetermined molar ratios. In one embodiment, the acid selected for the reaction is but not limited to citric acid and the base selected for the reaction is but not limited to a sodium carbonate. Any other acid-base mixture suitable to generate the CO₂ can also be used apart. As an example, the molar ratio of the citric acid and the sodium bicarbonate is provided in the below Table lfor generating CO₂.

Fig. 2 illustrates a portable device (10) for maintaining viability of the biological material according to an exemplary embodiment of the present disclosure. In an exemplary embodiment of the disclosure, the portable device (10) comprises of two compartments with a common air tight lid (16). An outer compartment (11b) is filled with acid-base reaction mixture (19) which consists of Hydrochloric acid and Sodium bicarbonate salt for generating CO₂ gas. An inner compartment (11a) is filled with a transportation medium or a cell culture medium (17) in which bio-cellular material or biological material (18) to be transported is stored. The outer compartment (11b) is provided with an injection port (rubber cork) through which a solvent (20) to make the acid-base mixture (19) to react is filled in the outer compartment (11b). In one embodiment, the portable device (10) is provided with two filters (15) attached at sides of the inner compartment (11a) through which theCO₂gas exchange from the outer compartment (l ib) to the inner compartment (11a) takes place. In one embodiment, the filters (15) are 0.2 μm syringe filters that would facilitate the transfer of sterile CO₂gas from the outer compartment (l ib) to the inner compartment (11a). In an embodiment of the disclosure, the portable device (10) of the present disclosure is designed in such a way that it mimics standard growth conditions as provided in the labs.

In one embodiment of the present disclosure a method of maintaining the viability of the biological material (18) is provided. Firstly, a desired quantity of suitable sterile medium or a cell culture media (17) is added directly using a pipette into the inner compartment (11a) in a sterile environment. A biological material(18) obtained from a source is placed inside the cell culture media (17) through opening of the inner compartment (11a) and thereafter a lid (16) is closed tightly. Secondly, a suitable solvent (20) is injected into the outer compartment (11b) through a port (16a). A suitable acid-base mixture (19) is provided into the outer compartment (11b) through a port (16a) and closed using a rubber cork. The injected solvent (20) contact with acid- base mixture (19) and initiates the reaction. The reaction generates CO₂ and the generated CO₂ is equally distributed inside the inner compartment (11a) through the plurality of filters (15) located at the junction of the inner compartment (11a) and the outer compartment (11b).

In one embodiment, calculated molar concentration of the acid-base mixture and the solvent generates the calculated amount of CO₂, which is about 5% for given size of the receptacle. In one embodiment, a plurality of heat packs is provided in the outer compartment and contacts the inner compartment for maintaining temperature of the culture media at about 37°C. The 5% of CO₂is stable for about 72 hours in an air tight sealed receptacle. Thus the 5% CO₂ and maintaining the temperature of the culture media at about 37 °C consecutively buffers the pH of the culture media for about 72 hours which in turn maintains the viability of the biological material for the specific period.

In one embodiment, depending on the size of the portable device (10) and transportation time, the quantity of acid-base mixture and number of heat packs can be increased to maintain the parameters like CO₂ and temperature for up to 120 hours or more.

In one embodiment, the portable device is packed in any commercial cold chain packing material and can be transported to a required destination.

Fig. 3illustrates a portable device (10) for maintaining viability of the biological material (18) according to another exemplary embodiment of the present disclosure. The reaction chamber (14) is configured in the outer compartment (11b) and the ports (16a) are also provided in the outer compartment (11b). In one embodiment, the heat pack (15) is provided in the base of the outer compartment (l ib) for maintaining the required temperature for the culture media (17) during transportation of the biological material (18) from one place to another place. The filters (15) are configured at the junction in the inner compartment (11a) and the outer compartment (11b).

Fig. 4illustratesa portable device (10) for maintaining viability of the biological material (18) according to another exemplary embodiment of the present disclosure. In one embodiment, the reaction chamber (14) can be configured in the inner compartment (11a).

In one embodiment, the acid-base mixture (19) can be in the form but not limited to a premixed powder form or gel form or capsule forms. In one embodiment, the capsule form of this acid-base mixture (19) is dropped into the culture media (17) directly. Capsules of controlled releasing nature can be used for this purpose.

Working example:

Workable culture media used for the different types of biological material that can be transported in the said device is provided in below table 2

Table 2

ur at, - , an macrop age ce s. , uta

The below table 3 provides the amount of media to be added for a specific volume of the device.

In one embodiment, a variety of chemical routes to carbon dioxide are known, such as the reaction between most acids and most metal carbonates.

For example, the reaction between hydrochloric acid and calcium carbonate (limestone or chalk) is depicted below:

2HC1+ CaCO3→ CaC12+ H2CO3; here, the carbonic acid (H2CO3) then decomposes to water and CO2.

Further, the following Table 4 represents the possible acid-bicarbonate/carbonate combinations that can generate CO₂.

The below Table 5 provides details of concentration of CO₂ generated at each different molar ratio of citric acid and sodium bicarbonate and its reaction time

The below Table 6 provide details of aspect of regularizing 3%, 4% or 5% of CO₂ as per the requirements of an experiment/study at each different molar ratio of citric acid and sodium bicarbonate.

Various methods used to check the viability of the cells at the end of 72 hours

1. Trypan Blue method

The Trypan Blue is the stain most commonly used to distinguish viable from nonviable cells. Viable cells exclude the dye, while nonviable cells absorb the dye and appear blue. Cells should be in suspension as single cells in buffered saline before counting. Aseptically withdraw a sample of the cell suspension and prepare 1:2, 1:5, 1: 10 or 1: 100 dilutions, as required in PBS. Dilute 1:5 in 0.5% Trypan Blue. The optimal concentration of cells for counting is 5 x 105 cells/ml (50-100 cells per large square) after dilution in the Trypan Blue solution. After being stained with Trypan Blue, the cells should be counted within 3 minutes, after that time the cells will begin to take up the dye. Using a Pasteur pipette, withdraw a small amount of the stained cell suspension and place the tip of the pipette onto the slot of a clean haemocytometer with cover slip. The cell suspension will pass under the cover slip by capillary action. Fill the opposite chamber with the second diluted sample. Do not overfill. Do not lift or move the cover slip. Place the haemocytometer on the stage of an inverted microscope. Adjust focus and power until a single counting square fills the field.

Calculate the average number of unstained cells in each quadrant, and multiply by 1x104 to find cells/ml.

The percentage of viable cells is the (number of viable cells) divided by the total number of (dead plus viable cells), multiplied by 100 % viable cells = 100 x (live cells)/ (dead+live cells).

2. Growth rate of the cells

Growth rate is assessed using a commercially prepared cell culture medium. The assay will be performed directly by culturing the biopsy by explant method or after the isolation of cells from biopsy and plated at 1 x 10⁴ cells/ml. Cultures will be plated in duplicate 35-mm-diameter petri dishes and incubated for 14 days at 37°C with 5 % CO₂ in a humidified atmosphere. Every alternate day medium will changed and cells are observed under microscope for growth rate and growth characteristics will be recorded.

3. Flow Cytometryassays

Cell surface markers will be analyzed to estimate the quality and stability of phenotype of the cells post processing by flow cytometry.

Advantages

In one embodiment, the combination of the inner compartment and the outer compartment in a portable manner separated by filters ensures regulated or controlled supply of sterile CO₂ from outer compartment into the inner compartment. The supply of the CO₂ is the critical factor in maintaining the viability of the biological material for about 72 hours. The supply of CO₂maintains the pH of the culture media in which biological material is stored.

In on embodiment, the device disclosed in the disclosure is economical and portable.

In one embodiment, the device is power independent since the heat packs in the device maintains the internal environment at 37°C for up to 72 hours without manual intervention.

In one embodiment, of the present disclosure once the acid-base reaction is completed, the released CO₂ is equally distributed throughout the receptacle. The amount of CO₂ generated is stable for 72 hours since the CO₂ is mainly a buffering component and its utilization for cell respiration is meagre. Hence continuous production and supply is not required for at least 72 hours. Loss of CO₂ during this period is compensated by controlled acid-base reaction.

In one embodiment, by providing 5% CO₂ using calculated molar concentration of acid-base reaction, the required amount of CO₂ in a portable handy device for a period of 72 hours is provided. The CO₂ pressure inside the receptacle regulates the amount of CO₂ dissolving in the culture media which in turn react with water molecules in the culture media and produce an intermediate compound as bicarbonate. The bicarbonate further dissociates into an acid and a base and that regulates the pH of the medium. Ambient temperature for the above said period and transportation induced vibration is taken care by the commercially available temperature controlled-cold chain packing material.

In one embodiment, the portable device replicates the standard growth conditions like lab based CO2 incubators and the method is an aseptic method of generation and introduction of CO2 and the controlled release of CO2.

In one embodiment, the biological materials such as cells are delivered in their active growth stage, i.e. the cells do not undergo stress due to change of preservation temperature and because of maintaining the internal environment for up to 72 hours without manual intervention.

In one embodiment, the device is handy and portable. Also, the device is simple in nature which would not require any special training to handle the device.

In one embodiment, the device is meets the manufacturing criterions such as medical grade plastic materials that meet FDA requirements, withstand extreme temperature, withstand extreme pressure, and chemically inert.

In one embodiment, the technology in the device is basically a miniaturized, portable CO₂ incubator which can be used for viable transportation of biological samples such as mammalian cells, tissues, organs or tissue engineered products. The device in this disclosure is unique in its portability, inexpensive, not cumbersome, does not require skilled labor, electricity and CO₂ cylinder.

In one embodiment, the CO₂ is fully oxidized and therefore is extremely stable by nature and in this invention the CO₂ generated at top will tend to come down as it is heavier than air. Thus it gets distributed to the inner compartment. Once it is in touch with the media (liquid) it will get dissolved. So there is no exclusive gas distribution system is required. Equivalents

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should typically be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, typically means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

We claim:

1. A portable device (10) for maintaining viability of a biological material, the device (10) comprising:

a receptacle (11) configured into an inner compartment (11a) and an outer compartment (11b) concentrically arranged into one another such that a gap (22) is created between the inner compartment (11a) and the outer compartment (11b), wherein the inner compartment (11a) contains a culture media and the biological material (18) inside a culture media (17);

a plurality of heat packs (12) placed in the gap (22) created between the inner compartment (11a) and the outer compartment (11b) to maintain temperature of the culture media (17) present in the inner compartment (11a); a reaction chamber (14) is configured in the portable device at a predetermined location, wherein the reaction chamber (14) comprises an acid- base mixture (19) and a predetermined solvent (20) for generating carbon dioxide by a chemical reaction inside the reaction chamber (14);

at least one filter (15) is configured to enable controlled supply of the carbon dioxide (21) generated from the reaction chamber (14) to the inner compartment (11a); and

at least one lid (16) to close the inner compartment (11a) and the outer compartment (11b), wherein the inner compartment (11a) is hermitically closed.

2. The portable device (10) as claimed in claim 1, wherein the reaction chamber (14) is connected to top surface of the inner compartment (11a) or bottom surface of the inner compartment (11a) or sides of the inner compartment (11a) or inside the inner compartment (11a).

3. The portable device (10) as claimed in claim 1, wherein the heat packs (12) are provided in between base of the outer compartment (11b) and base of the inner compartment (11a).

4. The portable device (10) as claimed in claim 1, wherein the reaction chamber (14) is configured to the at least one lid (16) closing the inner compartment (11b).

5. The portable device (10) as claimed in claim 1, wherein the lid (16) is configured with a plurality of ports (16a) for providing the acid-base mixture and the solvent to the reaction chamber (14).

6. The portable device (10) as claimed in claim 1, wherein the acid-base mixture is in form selected from a group comprising a premixed powder form or gel form or capsule forms.

7. The portable device (10) as claimed in claim 1, wherein the receptacle (11) is made of biocompatible material selected from a group comprising medical grade plastics, selected from a group comprising Polycarbonate, Polyetherimide, Polysulfone, PolyEtherEther-Ketone, Polyphenylene sulfide, Polyphenylsulfone, Polyacetal copolymer, Polyester, Polypropylene, Polytetrafluoroethylene, Poly(methyl methacrylate) (PMMA), Acrylonitrile butadiene styrene (ABS), Silicone.

8. The portable device (10) as claimed in claim 1 is configured to maintain the viability of the biological material (18) selected from a group comprising a cellular material, a tissue material and its combination thereof.

9. The portable device (10) as claimed in claim 1, wherein the heat packs (12) maintains temperature of the culture media (17) at about 37°C for a time of about 96-120 hours for maintain viability of the biological material (18).

10. The portable device (10) as claimed in claim 1, wherein the plurality of filters (15) is provided in junction of the reaction chamber (14) and the inner compartment (11a) or in the junction of the inner compartment (11a) and the outer compartment (1 lb).

11. A method for maintaining viability of a biological material using the portable device (10) as claimed in claim 1, the method comprising acts of:

providing an acid -base mixture (19) and the predetermined solvent (20) inside the reaction chamber (14) at predetermined molar concentration for generating carbon dioxide (21); and supplying the generated carbon dioxide (21) into the inner compartment (11a) in a controlled manner using the plurality of filters (15) for maintaining pH of the culture media (17), thereby to maintain viability of the biological material (18) for predetermined hours during transportation from one place to another place.

12. The method as claimed in claim 11, wherein the acid-base mixture (19) is an acid-carbonate mixture and the predetermined solvent (20) is water.

13. The method as claimed in claim 11, wherein the carbon dioxide (21) supplied to the inner compartment (11a) is in concentration ranging from 1% v/v to 10% v/v.