Patent Application
20140315303
Many techniques in biology and medicine, such as cell separation, flow cytometry, cell assays, and cell therapies are dependent on dissociating tissues to isolate individual cells. Processing tissue samples often involves multiple acts, such as mincing, washing, enzymatic digestion, dissociation, incubation, mixing, clump and debris removal, and concentration. In research laboratories, these acts are often done manually with the sample transferred from test tubes to test tubes in an open environment. Such manual processes require highly trained personnel and the manipulation of biological samples poses potential risks of contamination and infection.
Recently there has been interest in the research and medical communities to isolate cells from adipose tissues. Several techniques have been developed to safely remove portions of adipose tissues from a patient or an animal. For example, tumescent liposuction and water jet liposuction techniques have been widely used to remove fat tissue from patients. Adipose tissues contain adipose cells that store fat and other non-fat cells that maintain the tissues. The constituent cells of adipose tissues, their roles and their interactions are not completely understood, and are a subject of active academic and clinical research.
To study adipose tissues, it may be desirable to dissociate the tissue and isolate the constituent cells. The process may involve releasing the constituent cells, removing debris and unwanted cells, concentrating and enriching cells of interest, and washing the cells. Such process may be laborious and may require highly trained operators, expensive equipment setup, and a laboratory with proper biosafety measures. The multiple manipulation acts may also cause significant loss of cells of interest, making the isolation of rare and low prevalence cells difficult and unreliable. Furthermore, when human samples are used, the risks of cross-contamination and infection may be substantial.
It is therefore desirable to have a method for effective isolation of cells of interest from a tissue, and to have a device that makes isolation of cells from tissues easy and safe.
Bags comprising flexible plastic sheets are widely used to collect, process, and store biological tissue samples, such as peripheral blood, umbilical cord blood, blood components, plasma, bone marrow, lipoaspirates, etc. Bags have the advantage of being flexible and expandable, and are capable of changing their inner volumes to accommodate samples of different volumes. To facilitate sample processing, many individual bags are often fluidicly connected using external tubing to form a system. Such bag and tubing system have been widely used in sample processing, for example, blood fractionation, cell isolation, etc. However, as more processing acts are integrated, the bag and tubing systems quickly become cumbersome, hard to use, and difficult to manufacture. The systems become spaghetti-like and become prone to entanglement, with many components dangling off of each other. To use such devices, an operator needs a high level of training and a long period of hands-on time to set up the complicated devices. The operator also needs to pay extra attention to mount the various parts at the right places in the right order. Furthermore, these devices and systems may be difficult and costly to manufacture as multiple bags, components and tubing stretches often have to be made individually and then assembled. The assembling of such devices may be labor intensive, and may present risks of leakage and contamination, i.e., system failure. For clinical applications, the devices are often single use, and their reliability is important. The intensive labor and device failure risk is a major hurdle for these conventional spaghetti-like systems.
In accordance with an aspect of the present disclosure there is provided an apparatus for the processing of biological sample. The apparatus comprises a first sheet of material, a second sheet of material bonded to the first sheet of material, and a plurality of chambers defined between the first sheet of material and the second sheet of material, the plurality of chambers including a sample dissociation chamber including an inlet and an outlet; a waste collection chamber including an inlet in fluid communication with the outlet of the sample dissociation chamber, and a cell refinement chamber including an inlet in fluid communication with the sample dissociation chamber and an outlet.
In accordance with some embodiments the sample dissociation chamber further comprises a mesh filter.
In accordance with some embodiments the mesh filter comprises pores having a pore size of between 20 micrometers and 50 micrometers.
In accordance with some embodiments the apparatus further comprises a mesh filter included in the cell refinement chamber.
In accordance with some embodiments the sample dissociation chamber further comprises a first mesh filter comprising pores having a first pore size, and wherein the cell refinement chamber further comprises a second mesh filter comprising pores having a second pore size.
In accordance with some embodiments the second pore size is smaller than the first pore size.
In accordance with some embodiments the apparatus further comprises a means to control fluid connection between the sample dissociation chamber, the waste collection chamber and the cell refinement chamber.
In accordance with some embodiments the means to control fluid connection comprises a stopcock.
In accordance with some embodiments the apparatus further comprises a flow control device configured to introduce at least one of a rinsing solution and a dissociation solution into the sample dissociation chamber and having an outlet in fluid communication with the tissue dissociation chamber.
In accordance with some embodiments the apparatus further comprises means for applying pressure to one of the sample disassociation chamber and the cell refinement chamber. The means may be disposed between the first sheet of material and the second sheet of material. The means may be disposed about the first sheet of material and/or the second sheet of material.
In accordance with some embodiments the apparatus further comprises a downstream processing apparatus in fluid communication with the outlet of the cell refinement chamber and including at least one microfluidic device configured to separate a fluid output from the cell refinement chamber into a first solution having a first concentration of one or more cells of interest and a second solution having a concentration of the one or more cells of interest which is less than that of the first solution.
In accordance with an aspect of the present disclosure there is provided an apparatus for the processing of biological tissue. The apparatus comprises a first sheet of material, a second sheet of material bonded to the first sheet of material and a plurality of chambers defined between the first sheet of material and the second sheet of material, the plurality of chambers including a tissue dissociation chamber including an inlet, a first outlet, a second outlet, and first mesh filter, a waste collection chamber including an inlet in fluid communication with the first outlet of the tissue dissociation chamber, and one of a cell refinement chamber and a sample collection chamber including an inlet in fluid communication with the second outlet of the tissue dissociation chamber.
In accordance with some embodiments the apparatus further comprises a measuring chamber including an outlet in fluid communication with the inlet of the tissue dissociation chamber.
In accordance with some embodiments each of the tissue dissociation chamber, the waste collection chamber, the cell refinement chamber, and the measuring chamber are defined between the first sheet of material and the second sheet of material.
In accordance with some embodiments the apparatus further comprises a clump reduction chamber in fluid communication between the tissue dissociation chamber and the one of the cell refinement chamber and the sample collection chamber.
In accordance with some embodiments each of the tissue dissociation chamber, the waste collection chamber, the cell refinement chamber, and the clump reduction chamber are defined between the first sheet of material and the second sheet of material.
In accordance with some embodiments the apparatus further comprises a second mesh filter included in the tissue dissociation chamber downstream of the first mesh filter.
In accordance with some embodiments the apparatus further comprises a mesh filter included in the one of the cell refinement chamber and the sample collection chamber.
In accordance with some embodiments the apparatus further comprises a flow control device configured to introduce one of a rinsing solution and a dissociation solution into the tissue dissociation chamber and having an outlet in fluid communication with the tissue dissociation chamber.
In accordance with some embodiments the apparatus further comprises means for applying pressure to one of the tissue disassociation chamber and the one of the cell refinement chamber and the sample collection chamber.
In accordance with some embodiments the apparatus further comprises a downstream processing apparatus in fluid communication with an outlet of the one of the cell refinement chamber and the sample collection chamber and including a microfluidic device configured to separate a fluid output from the one of the cell refinement chamber and the sample collection chamber into a first solution having a first concentration of one or more cells of interest and a second solution having a concentration of the one or more cells of interest which is less than that of the first solution.
In accordance with an aspect of the present disclosure there is provided a sterile and substantially isolated tissue processing system. The system comprises a tissue processing chamber including an inlet, an outlet, and at least one mesh filter disposed between the inlet of the tissue processing chamber and the outlet of the tissue processing chamber, a waste collection chamber included in a same enclosure as the tissue processing chamber, the waste collection chamber including an inlet in fluid communication with the outlet of the tissue processing chamber, and one of a debris removal chamber including a debris removal mechanism, and a sample collection chamber included in the same enclosure as the tissue processing chamber and in fluid communication with the second outlet of the tissue processing chamber.
In accordance with an aspect of the present disclosure there is provided substantially isolated tissue processing system. The system comprises a tissue processing chamber including an inlet, a first outlet, a second outlet, and at least one mesh filter disposed between the inlet of the tissue processing chamber and the first outlet of the tissue processing chamber, a waste collection chamber included in a same enclosure as the tissue processing chamber, the waste collection chamber including an inlet in fluid communication with the first outlet of the tissue processing chamber, and one of a debris removal chamber including a debris removal mechanism, and a sample collection chamber included in the same enclosure as the tissue processing chamber and in fluid communication with the second outlet of the tissue processing chamber.
In accordance with some embodiments the system further comprises a fluid volume measuring chamber in the same enclosure as the tissue processing chamber and including an inlet and an outlet in fluid communication with the inlet of the tissue processing chamber.
In accordance with some embodiments the inlet of the fluid volume measuring chamber and the outlet of the fluid volume measuring chamber each include check valves.
In accordance with some embodiments the first outlet and the second outlet comprise outlets of a stopcock in fluid communication with the tissue processing chamber.
In accordance with an aspect of the present disclosure there is provided a method of processing a tissue sample in a tissue processing system. The method comprises introducing a fluid including a tissue sample to be processed into a fluid volume measuring chamber through an inlet port of the fluid volume measuring chamber, transferring a pre-determined volume of the fluid from the fluid volume measuring chamber to a tissue processing chamber through an outlet port of the fluid volume measuring chamber and an inlet port of the tissue processing chamber, treating the tissue sample in the tissue processing chamber and releasing a sample of cells from the tissue processing chamber through a second outlet of the tissue processing chamber an into a sample storage chamber included in a same enclosure as the tissue processing chamber through an inlet of the sample storage chamber.
In accordance with some embodiments the method further comprises closing the inlet port of the fluid volume measuring chamber and opening the outlet port of the fluid volume measuring chamber subsequent to introducing the fluid including a tissue sample to be processed into the fluid volume measuring chamber and prior to transferring the pre-determined volume of the fluid from the fluid volume measuring chamber to the tissue processing chamber.
In accordance with some embodiments the method further comprises retaining a sample of cells within the tissue processing chamber and transferring a waste fluid through a mesh filter included in the tissue processing chamber and a first outlet of the tissue processing chamber into a waste collection chamber included in the same enclosure as the tissue processing chamber through an inlet of the waste collection chamber.
In accordance with some embodiments the method further comprises extracting the sample of cells from the tissue processing system.
In accordance with some embodiments treating the tissue sample in the tissue processing chamber comprises introducing a tissue cleaning solution into the tissue processing chamber.
In accordance with some embodiments treating the tissue sample in the tissue processing chamber further comprises introducing a tissue dissociating solution into the tissue processing chamber.
In accordance with an aspect of the present disclosure there is provided a method of processing a sample in a tissue processing system. The method comprises introducing a sample to be processed into a tissue processing chamber through an inlet port of the tissue processing chamber, treating the sample in the tissue processing chamber, and releasing cells from the tissue processing chamber through an outlet of the tissue processing chamber into a sample storage chamber included in a same enclosure as the tissue processing chamber through an inlet of the sample storage chamber.
In accordance with some embodiments the method further comprises extracting the sample of cells from the tissue processing system.
In accordance with some embodiments the method further comprises processing the extracted sample of cells in a downstream processing apparatus in fluid communication with an outlet of the sample storage chamber and including at least one microfluidic device configured to separate the extracted sample of cells into a first solution having a first concentration of one or more cells of interest and a second solution having a concentration of the one or more cells of interest which is less than that of the first solution.
In accordance with an aspect of the present disclosure there is provided a method of processing a sample in a tissue processing system. The method comprises introducing a sample to be processed into a tissue processing chamber through an inlet port of the tissue processing chamber, treating the sample in the tissue processing chamber, and transferring cells from the tissue processing chamber through an outlet of the tissue processing chamber into a sample storage chamber included in a same enclosure as the tissue processing chamber through an inlet of the sample storage chamber.
In accordance with some embodiments treating the sample comprises dissociating the sample.
In accordance with some embodiments treating the sample comprises removing excess fluids from the sample.
In accordance with some embodiments treating the sample comprises washing the sample using a rinsing solution.
In accordance with some embodiments treating the sample comprises washing the sample using a rinsing solution and dissociating the sample using a dissociation solution comprising at least one enzyme.
In accordance with some embodiments the method further comprises the removal of debris using a mesh filter included in the sample storage chamber.
In accordance with some embodiments the mesh filter has a pore size of between 15 micrometers and 50 micrometers.
In accordance with some embodiments the method further comprises retaining a sample of cells within the tissue processing chamber and transferring a waste fluid through a mesh filter included in the tissue processing chamber and a first outlet of the tissue processing chamber into a waste collection chamber included in the same enclosure as the tissue processing chamber through an inlet of the waste collection chamber.
In accordance with some embodiments the method further comprises enriching the cells for a target cell population using a microfluidic device.
In accordance with some embodiments the method further comprises extracting the cells from the tissue processing system.
In accordance with some embodiments the method further comprises processing cells in a downstream processing apparatus in fluid communication with an outlet of the sample storage chamber and including at least one microfluidic device configured to separate the cells into a first solution having a first concentration of one or more cells of interest and a second solution having a concentration of the one or more cells of interest which is less than that of the first solution.
In accordance with some embodiments treating the tissue sample in the tissue processing chamber comprises introducing a tissue cleaning solution into the tissue processing chamber.
In accordance with some embodiments treating the tissue sample in the tissue processing chamber further comprises introducing a tissue dissociating solution into the tissue processing chamber.
In accordance with some embodiments the cells extracted are adipose derived stem cells.
In accordance with some embodiments the cells extracted are mesenchymal stem cells.
In accordance with some embodiments the cells extracted are stem cells.
In accordance with some embodiments the cells extracted are pancreatic islet cells.
In accordance with some embodiments the cells extracted are bacteria.
In accordance with some embodiments the cells extracted are stromal vascular fraction cells.
In accordance with some embodiments the cells extracted are stem cells derived from an umbilical cord.
In accordance with some embodiments the cells extracted are yeasts.
In accordance with some embodiments the cells extracted are parasites.
In accordance with some embodiments the cells extracted are a foodborne pathogen.
In accordance with an aspect of the present disclosure there is provided a substantially isolated tissue processing system. The system comprises a fluid volume measuring chamber including an inlet and an outlet, a tissue processing chamber included in a same enclosure as the fluid volume measuring chamber, the tissue processing chamber including an inlet in fluid communication with the outlet of the fluid volume measuring chamber, a first outlet, a second outlet, and at least one mesh filter disposed between the inlet of the tissue processing chamber and the first outlet of the tissue processing chamber, a waste collection chamber included in the same enclosure as the fluid volume measuring chamber and tissue processing chamber, the waste collection chamber including an inlet in fluid communication with the first outlet of the tissue processing chamber, and one of a debris removal chamber including a debris removal mechanism, and a sample collection chamber included in the same enclosure as the fluid volume measuring chamber and tissue processing chamber and in fluid communication with the second outlet of the tissue processing chamber.
In accordance with some embodiments the inlet of the fluid volume measuring chamber and the outlet of the fluid volume measuring chamber each include check valves.
In accordance with an aspect of the present disclosure there is provided a substantially isolated tissue processing system. The system comprises a sample washing and dissociation chamber, including three inlet ports, a first outlet port and a second outlet port, and a mesh disposed between the three inlet ports and the first and second outlet ports, a clump reduction chamber comprising an inlet connector in fluid connection with the first outlet port of the sample washing and dissociation chamber, an outlet, and a mesh disposed between the inlet connector and the outlet, a reservoir for isolated cells and further debris removal having an inlet in fluid communication with the outlet of the clump reduction chamber, and a waste solution collection chamber, having an inlet in fluid communication with the second outlet port of the sample washing and dissociation chamber.
In accordance with some embodiments each of the sample washing and dissociation chamber, the clump reduction chamber, the reservoir, and the waste solution collection chamber included in a same sealed packaging.
In accordance with an aspect of the present disclosure there is provided a method of processing a tissue sample in a tissue processing system. The method comprises introducing a fluid including a tissue sample to be processed into a fluid volume measuring chamber through an inlet port of the fluid volume measuring chamber while an outlet port of the fluid volume measuring chamber is closed, closing the inlet port of the fluid volume measuring chamber, opening the outlet port of the fluid volume measuring chamber, transferring a pre-determined volume of the fluid from the fluid volume measuring chamber to a tissue processing chamber located within a same enclosure as the fluid volume measuring chamber through the outlet port of the fluid volume measuring chamber and an inlet port of the tissue processing chamber, treating the tissue sample in the tissue processing chamber, and releasing the sample of cells from the tissue processing chamber through a second outlet of the tissue processing chamber an into a sample storage chamber included in the same enclosure as the fluid volume measuring chamber and tissue processing chamber through an inlet of the sample storage chamber.
In some embodiments the method further comprises retaining a sample of cells within the tissue processing chamber and transferring a waste fluid through a mesh filter included in the tissue processing chamber and a first outlet of the tissue processing chamber into a waste collection chamber included in the same enclosure as the fluid volume measuring chamber and tissue processing chamber through an inlet of the waste collection chamber.
In some embodiments the method further comprises extracting the sample of cells from the tissue processing system.
In accordance with an aspect of the present disclosure there is provided a method of processing a tissue sample in a tissue processing system. The method comprises introducing a tissue sample processing solution into a fluid volume measuring chamber through an inlet port of the fluid volume measuring chamber while an outlet port of the fluid volume measuring chamber is closed, closing the inlet port of the fluid volume measuring chamber, opening the outlet port of the fluid volume measuring chamber, transferring a pre-determined volume of the solution from the fluid volume measuring chamber to a tissue processing chamber located within a same enclosure as the fluid volume measuring chamber through the outlet port of the fluid volume measuring chamber and a first inlet port of the tissue processing chamber, introducing a tissue sample to be treated into the tissue processing chamber through a second inlet port of the tissue processing chamber, treating the tissue sample in the tissue processing chamber, releasing the sample of cells from the tissue processing chamber through a second outlet of the tissue processing chamber an into a debris removal chamber included in the same enclosure as the fluid volume measuring chamber and tissue processing chamber through an inlet of the debris removal chamber, and removing undesired cells from the sample of cells in the debris removal chamber to form a purified cell sample.
In some embodiments the method further comprises retaining a sample of cells within the tissue processing chamber and transferring a waste fluid through a mesh filter included in the tissue processing chamber and a first outlet of the tissue processing chamber into a waste collection chamber included in the same enclosure and the fluid volume measuring chamber and tissue processing chamber through an inlet of the waste collection chamber.
In some embodiments the method further comprises extracting the purified cell sample from the tissue processing system.
In accordance with an aspect of the present disclosure there is provided an apparatus for isolation of non-fat cells from an adipose tissue sample. The apparatus comprises a first sheet of material, a second sheet of material bonded to the first sheet of material, and a plurality of chambers defined between the first sheet of material and the second sheet of material, the plurality of chambers including a sample dissociation chamber including an inlet and an outlet, a waste collection chamber including an inlet in fluid communication with the outlet of the sample dissociation chamber, and a cell refinement chamber including an inlet in fluid communication with the sample dissociation chamber and an outlet.
In accordance with some embodiments the sample dissociation chamber further comprises a mesh filter comprising pores having a pore size of between 70 μm and 300 μm.
In accordance with some embodiments the apparatus further comprises a mesh filter included in the cell refinement chamber comprising pores having a pore size of between 20 μm and 50 μm.
In accordance with some embodiments the sample dissociation chamber further comprises a first mesh filter comprising pores having a first pore size, and wherein the cell refinement chamber further comprises a second mesh filter comprising pores having a second pore size, wherein the second pore size is smaller than the first pore size.
In accordance with some embodiments the apparatus further comprises a means to control fluid connection between the sample dissociation chamber, the waste collection chamber and the cell refinement chamber.
In accordance with some embodiments the means to control fluid connection comprises a stopcock.
In accordance with some embodiments the apparatus further comprises a flow control device configured to introduce at least one of a rinsing solution and a dissociation solution into the sample dissociation chamber and having an outlet in fluid communication with the sample dissociation chamber.
In accordance with some embodiments the apparatus further comprises means for applying pressure to one of the sample disassociation chamber and the cell refinement chamber.
In accordance with some embodiments the apparatus further comprises a downstream processing apparatus in fluid communication with the outlet of the cell refinement chamber and including at least one microfluidic device configured to separate a fluid output from the cell refinement chamber into a first solution having a first concentration of one or more cells of interest and a second solution having a concentration of the one or more cells of interest which is less than that of the first solution, wherein the cells of interest comprise non-fat cells isolated from an adipose tissue sample.
In accordance with an aspect of the present disclosure there is provided a sterile and substantially isolated adipose tissue processing system. The system comprises a tissue processing chamber including an inlet, an outlet, and at least one mesh filter disposed between the inlet of the tissue processing chamber and the outlet of the tissue processing chamber, a waste collection chamber included in a same enclosure as the tissue processing chamber, the waste collection chamber including an inlet in fluid communication with the outlet of the tissue processing chamber, and one of a debris removal chamber including a debris removal mechanism, and a sample collection chamber included in the same enclosure as the tissue processing chamber and in fluid communication with the tissue processing chamber.
In accordance with an aspect of the present disclosure there is provided a method of processing an adipose tissue sample in a tissue processing system. The method comprises introducing an adipose tissue sample to be processed into a first chamber through an inlet port of the first chamber, treating the adipose tissue sample in the first chamber, and transferring cells from the first chamber through an outlet of the first chamber into a second chamber included in a same enclosure as the first chamber through an inlet of the second chamber.
In accordance with some embodiments treating the adipose tissue sample comprises dissociating the adipose tissue sample.
In accordance with some embodiments treating the adipose tissue sample comprises removing excess fluids from the adipose tissue sample in the first chamber.
In accordance with some embodiments treating the adipose tissue sample comprises washing the adipose tissue sample in the first chamber using a rinsing solution.
In accordance with some embodiments treating the adipose tissue sample comprises washing the adipose tissue sample in the first chamber using a rinsing solution and dissociating the adipose tissue sample in the first chamber using a dissociation solution comprising at least one enzyme.
In accordance with some embodiments the dissociation solution comprises collagenase.
In accordance with some embodiments the dissociation solution comprises collagenase, deoxyribonuclease and hyaluronidase.
In accordance with some embodiments dissociating the adipose tissue sample using a dissociation solution occurs at about 37 degrees Celsius.
In accordance with some embodiments the method further comprises the removal of debris using a mesh filter included in the second chamber.
In accordance with some embodiments the mesh filter has a pore size of between 15 micrometers and 100 micrometers.
In accordance with some embodiments the method further comprises retaining the sample within the first chamber and transferring a waste fluid through a mesh filter included in the first chamber and a first outlet of the first chamber into a third chamber included in the same enclosure as the first chamber through an inlet of the third chamber.
In accordance with some embodiments the method further comprises enriching for non-fat cells population using a microfluidic device.
In accordance with some embodiments the non-fat cells comprise stem cells.
In accordance with some embodiments the method further comprises harvesting cells from the tissue processing system.
In accordance with some embodiments the method further comprises processing the cells in a downstream processing apparatus in fluid communication with an outlet of the second chamber and including at least one microfluidic device configured to separate the cells into a first solution having a first concentration of non-fat cells and a second solution having a concentration of non-fat cells which is less than that of the first solution.
In accordance with some embodiments the cells harvested are stromal vascular fraction cells.
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. All drawings should be considered schematic unless otherwise indicated. In the drawings:
This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
The term “sample” as used herein may include a tissue, an animal tissue, a connective tissue, a muscle tissue, a nervous tissue, an epithelial tissue, a solid tumor tissue, a placenta tissue, an umbilical cord tissue, a tissue containing stem cells, a pancreatic tissue, a brain tissue, a heart tissue, an adipose tissue, a solid tissue, pancreatic islets, a pancreatic tissue, a liver tissue, a tissue containing progenitor cells and/or stem cells, a skin tissue, a ligament tissue, a bone tissue, a mesenchymal tissue, a tissue containing cells of interest, a tissue containing hepatocytes, a tissue containing fibroblasts, a tissue containing keratinocytes, a tissue containing chondrocytes, a tissue containing cardiomyocytes, a tissue containing oocytes, a tissue containing nerve cells, an umbilical cord, a tissue from an umbilical cord, cells imbedded in a matrix, cells embedded in an extracellular matrix, plant tissues, and other tissue pieces of biological origin, whether dead or alive. The term “sample” as used herein may also include a multi cell organism, a complete organism, parasites, biomass, a food sample, hamburger patties, beef, lamb, chicken, pork, turkey, shell fish, fish, poultry, ground beef, ground meat, ground chicken, ground turkey, ground pork, ground lamb, hot dogs, corn dogs, mixed meat, candy bars, and peanut butter.
The term “microfluidic device” as used herein may refer to a device having at least one fluidic channel formed on substantially one surface, which may be substantially planar or curved, and at least one lateral channel dimension smaller than about 1 mm. The lateral channel dimension may be for example, the width or the depth of the channel.
It has been found to be desirable to have a method for effective isolation of cells of interest from a tissue, and to have a device that makes isolation of cells from tissues easy and safe. For research and clinical applications, it has been found to be desirable that the multiple acts of processing tissue samples, such as those listed above, be streamlined so that human error can be minimized. Further, it has been found to be important that tissue samples are processed in a substantially “isolated” environment, where barriers are provided to isolate the samples from direct physical contact, or fluid contact, for example, through unfiltered air flow, with the external environment and/or operating personnel to minimize or avoid contamination and infection risks. It has also been found to be desirable to have a system and device for tissue processing where many components and compartments are integrated as one piece to provide a substantially isolated environment to the sample. It is preferable that a system and device for tissue processing is easy to use, easy to manufacture, and has low risk of failure. For many clinical and research applications, it may also be preferable that any parts of the device and system in direct contact with tissue samples are sterile and disposable.
Aspects and embodiments of the present disclosure provide a method for isolating certain constituent cell populations from a tissue sample. Other aspects and embodiments of the present disclosure provide a device for enabling the method for isolating certain constituent cell populations from a tissue sample in an integrated, streamlined, safe, and easy-to-use manner.
Aspects and embodiments of the present disclosure provide an integrated device comprising multiple compartments for tissue sample processing, which may include, but are not limited to, compartments configured and arranged for sample collection, washing, stratification, mixing, heating, cooling, filtering, digestion, storage, fluid transfer and manipulation, cell labeling, sample treatment, dissociation, waste fluid collection, clump removal, debris removal, cell concentration, cell enrichment, cell isolation, cell incubation, growth, culturing, differentiation, expansion, etc. Integrated devices in accordance with embodiments of the present disclosure may also include valving for the purpose of, for example, controlling fluid flow between compartments. Such devices may be useful for integrating and streamlining multiple acts of tissue sample processing, for example, isolating cells from tissues, and may facilitate multiple functions such as enzymatic digestion, tissue dissociation, washing, waste liquid collection, debris removal, cell concentration, labeling using antibodies, labeling using magnetic beads, cell expansion, etc. Such devices may be particularly useful for applications where safety, ease of use, and ease of manufacturing are important. Some aspects and embodiments of the present disclosure comprise methods for using such device.
One embodiment of the presently disclosed method for isolating cells from a tissue comprises, but is not limited to, dissociating the tissue, releasing the constituent cells, collecting the released cells, and removing tissue debris. The method may further comprise a tissue clean up act before tissue dissociation. The tissue clean up act may comprise removing or draining unwanted or excess fluids from the tissue sample. Such unwanted fluids may include blood, body fluids, saline solutions, tumescent solution, anesthetics, components that may interfere with potential downstream use of the cells, etc. The tissue clean up act may further comprise rinsing or washing the tissue using a rinsing solution. The method may further include one or more acts to enrich or purify the released cells. In addition, the method may further comprise collection of waste fluids from the process. Another embodiment of the presently disclosed method for isolating cells from a tissue comprises, but is not limited to, removing excess fluids from a tissue, dissociating the tissue and releasing the constituent cells, and removing unwanted cells and debris. The method may further comprise one or more of washing the tissue sample, concentrating the cells of interest, washing the cells of interest, and performing immuno-separation using, for example, antibodies. In one embodiment, concentrating and/or washing the cells of interest may be performed using at least one microfluidic device. In another embodiment, one or more acts may employ centrifugation. In yet another embodiment, one or more acts may be performed utilizing hollow fibers.
For example, in one embodiment of the present disclosure there is provided a method for isolating a non-fat cell population from an adipose tissue. The method includes, but is not limited to, removing excess fluids from the adipose tissue, washing the adipose tissue with a buffer solution, dissociating the tissue using, for example, ultrasound or a dissociation solution containing enzymes, removing fat cells, free oil, matrix fibers and tissue debris, reducing red blood cells, and enriching cells of interest. The cell enrichment act may be achieved using a centrifuge, a filter, or a microfluidic device. The method may further comprise one or more of lymphocyte reduction, cell washing, and immuno-separation. The acts of removing excess fluids from the adipose tissue, washing the adipose tissue with a buffer solution, dissociating the tissue, removing fat cells, free oil, matrix fibers and tissue debris, and cell washing may be performed using, for example, settling under gravity, centrifugation, and/or a strainer comprising a mesh filter.
A flow chart of one embodiment of a method for isolating a non-fat cell population from an adipose tissue is shown in
In another embodiment of the present disclosure, the excess fluid removal act may be performed by putting the sample in a container having an outlet and then draining the excess fluid without using a strainer. In one embodiment, the container may further comprise means for fluid control, for example, a pinch valve. To perform the removal of excess fluids, the excess fluids may be drained through the outlet, and when the sample approaches the outlet, the outlet can be closed off using the fluid control means. The outlet may have a size smaller than the tissue sample or a large size that allows for the tissue sample to pass through. The act may be performed manually or with the aid of a sensor, for example, an optical sensor or an infrared sensor, which detects the sample with respect to the outlet. A wash solution may be added to the tissue sample to wash or rinse the sample, and the excess fluid removal act may be repeated to clean up the tissue sample. The second act of the method shown in
After adding the dissociation solution, the tissue may be incubated at a certain temperature, for example, about 37 degrees Celsius, for a certain period of time, for example, from about 5 minutes to about 30 hours. During incubation, the tissue and the dissociation solution may be mixed intermittently and/or continuously to facilitate efficient reaction. The tissue dissociation act or the incubation act may be performed at 37 degrees Celsius for between about 10 minutes to about 120 minutes, for example, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, or about 120 minutes, with intermittent gentle agitation of the tissue sample in the dissociation solution, where the agitation occurs more frequent than every 3 minutes, for example, every second, every 2 seconds, every 3 seconds, every 5 seconds, every 10 seconds, every 20 seconds, every 30 seconds, every 45 seconds, every 60 seconds, every 90 seconds, every 120 seconds, or every 180 seconds.
At the end of the dissociation act, metal ion chelators such as ethylenediaminetetraacetic acid (EDTA) may be added to sequester metal ions and halt the activities of the enzymes in the dissociation solution, and the temperature may be lowered to between about 4 degrees Celsius and 30 degrees Celsius, for example, room temperature, about 25 degrees Celsius, about 22 degrees Celsius, about 18 degrees Celsius, about 15 degrees Celsius, about 12 degrees Celsius, about 8 degrees Celsius, or about 4 degrees Celsius. The temperature may be kept at around room temperature, for example, around 25 degrees Celsius, or between 18 degrees Celsius and 28 degrees Celsius, after incubation. In another embodiment, plasma, platelet enriched plasma, or serum may be added to inhibit the enzymes in the dissociation solution after the dissociation act.
The third act of the method of
The fourth, fifth and sixth acts of the method of
The sixth act of the embodiment shown in
In one embodiment of the present disclosure, a method comprises pre-conditioning of tissue, dissociation of tissue, and refinement of released cells. A flow chart of this embodiment of this method is shown in
In one embodiment, the tissue dissociation act (act 220) comprises incubating the tissue sample in a dissociation solution comprising at least one enzyme, for example, collagenase, protease, proteinase, neutral protease, elastase, hyaluronidase, lipase, trypsin, papain, liberase, DNase, deoxyribonuclease, pepsin, or a combination thereof, at a temperature suitable for enzyme digestion, for example, between about 32 degrees Celsius and about 38 degrees Celsius, for a duration of between about 3 minutes and 20 hours. In another embodiment, the tissue dissociation act comprises passing an ultrasound wave through the tissue sample. Cells are released from the tissue sample during the tissue dissociation act.
The refinement of released cell (act 230) may comprise cell concentration, cell washing, cell separation, cell isolation, debris removal, non-target cell removal, red blood cell reduction, or a combination of acts thereof, using a filter, a mesh, a hollow fiber, an antibody, a microfluidic device, or a centrifuge. For many applications, such as point of care applications and field applications, it may be desirable to perform the entire method in the present disclosure in within a short period of time, for example, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, or 120 minutes.
It is appreciated that many acts and embodiments disclosed herein may also be used for other tissue processing applications, and embodiments of the present disclosure are not limited to isolating non-fat cells from adipose tissues. For example, the embodiments may be applied to dissociation of solid tumors, placenta tissue, tissue containing stem cells, pancreatic tissue, brain tissue, heart tissue, pancreatic islets, pancreatic tissue, liver tissue, tissues containing progenitor cells and/or stem cells, skin tissue, ligament tissue, connective tissue, mesenchymal tissue, tissue containing cells of interest, or other tissue pieces. Many acts and embodiments disclosed herein for isolating a non-fat cell population from an adipose tissue may also be used in research studies and pharmacological test systems. Isolation of cells, for example, hepatocytes, fibroblasts, keratinocytes, chondrocytes, cardiomyocytes, oocytes, nerve cells and stem cells, as well as in vitro cultivated tissues may be performed for the physiological, metabolic and functional studies or for drug testing in pharmacological test systems. Many acts and embodiments disclosed herein for isolating a non-fat cell population from an adipose tissue may also be used in tissue engineering for transplantation. Cells obtained using the disclosed methods and/or devices herein may be cultured in vitro to form in vitro cultured tissues for transplantation. For example, hepatocytes may be isolated and transplanted to cure chronic liver diseases or to substitute liver function after acute organ failure. Chondrocytes may be isolated and cultivated to replace damaged cartilage. Dermal fibroblasts and keratinocytes may be isolated to build up three-dimensional skin transplants to treat burning injuries, diabetic or other ulcers. Tumor cells from various tissues may be isolated and fused with dendritic cells for tumor immunotherapy. Adipose-derived stem cells may be isolated to generate functional cells and tissue.
It is also appreciated that many acts and embodiments disclosed herein may also be used to obtain pancreatic islet cells from pancreatic tissue. Transplantation of pancreatic islets of Langerhans is a promising viable treatment option for patients with type-1 diabetes. Patients who need to have pancreatic surgery, including complete removal of the pancreas due to chronic inflammation, may also be transplant candidates. Transplantation of islets may restore some of the functions of the removed organ. Clinical studies have shown that a number of diabetic patients who had received injections of isolated islets into the liver were insulin independent for several years. Human islets for transplantation may be isolated from donor pancreases using embodiments of the methods and/or the devices disclosed herein. Since availability of donor organs is often limited, high yields of isolated islets per organ is highly desired. Collagenase, or collagenase in combination with neutral protease, other enzymes and/or ultrasound, may be used to dissociate the supporting matrix of the pancreas.
In another embodiment of the present disclosure, solid tumors removed from patients during surgery procedures may be dissociated and prepared to obtain tumor cells for molecular testing, genetic testing, drug testing and/or other testing and analysis to acquire information that may alter, influence, benefit, determine, or optimize treatment decisions. Cancer cells or tumor cells processed using the methods and/or the devices disclosed herein can be used directly for proteomics applications such as identification of biomarkers, testing against biomarkers, and mass spectrometry analysis, for RNA-based applications such as microarray hybridization and genetic analysis, for testing against drugs, and/or for cytology applications such as flow cytometry analysis and immunophenotyping. The cells that may be prepared from the present disclosure includes breast cancer cells, kidney cancer cells, liver cancer cells, lung cancer cells, nasopharyngeal cancer cells, ovarian cancer cells, and prostate cancer cells. Cells can also be used to purify proteins to test antibody-based cancer therapies. Alternatively, cells can be used to establish primary cell lines.
It is further appreciated that many acts and embodiments disclosed herein may also be used for food safety applications, such as the dissociation of food, for example, hamburger patties, beef, lamb, chicken, pork, turkey, shell fish, fish, poultry, ground beef, ground meat, ground chicken, ground turkey, ground pork, ground lamb, hot dogs, corn dogs, mixed meat, candy bars, peanut butter, etc. for food safety or other applications. Potential bacteria, viruses, yeast, parasites, and other foodborne pathogens, for example, Staphylococcus Aureus, Listeria Monocytogenes, Clostridium Botulinum, Salmonella, Escherichia coli, E. Coli O157:H7, etc., may be released and enriched from the dissociated food samples using aspects of the embodiments disclosed herein. The pathogens may then be detected using cell culture techniques, antibody based techniques such as lateral flow assays and enzyme-linked immunosorbent assay (ELISA), molecular techniques such as polymerase chain reaction (PCR), fluorescence in situ hybridization using peptide nucleic acid probes (PNA FISH), and enzymatic amplification or other techniques. Embodiments of the methods disclosed herein as methods for food sample preparation enable sensitive detection of pathogens, because pathogens embedded in the food sample may be released, enriched, and detected.
One embodiment of the present disclosure is a sample processing device shown schematically in
The rinsing solution may enter the conditioning chamber via a flow control device 330, for example, a peristaltic pump, which controls the volume of the rinsing solution being added to the conditioning chamber. The flow control device may comprise at least one valve and varying-volume container. For example, the flow control device may comprise a stopcock 370 and a syringe 380 as schematically shown in
Another embodiment of the present disclosure is a sample processing device shown schematically in
The cell refinement device is connected to the dissociation chamber. In some embodiments, the sample processing device further comprises a valve 435 between the dissociation chamber and the cell refinement device. The valve may be closed to allow incubation of the sample with the dissociation solution, and opened to allow the released cells to enter the cell refinement device.
The cell refinement device is configured to receive the released cells from the dissociation chamber and to refine the released cells. In some embodiments, the cell refinement device comprises a chamber fluidicly connected to the dissociation chamber via an inlet 445, an outlet 455 for harvesting refined cells 440, and a strainer configured to remove large debris in the dissociated sample. The strainer may comprise a filter having pore sizes of between about 10 μm and about 100 μm, for example, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 50 μm, about 70 μm, or about 100 μm. The strainer may also comprise a mesh with pore sizes of between about 20 μm and about 60 μm, for example, about 20 μm, about 22 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 50 μm, and about 60 μm.
Another embodiment of the present disclosure is a sample processing device shown schematically in
In some embodiments of the present disclosure, an adipose tissue sample is pre-warmed to a certain temperature, for example, 37 degrees Celsius before being loaded into the First Chamber 510. The pre-warming as a treatment of the adipose tissue sample may shorten the time required for processing the tissue sample. In other embodiments of the present disclosure, an adipose tissue sample is photo-activated using a light having an average wavelength of between 300 nm and 700 nm, before being loaded into the First Chamber 510. Photo-activation may increase the potency of the cells in the tissue sample. In yet another embodiment of the present disclosure, an adipose tissue sample is treated using an acoustic wave, i.e. a sound wave to loosen up the tissue rendering the tissue sample easier to dissociate, before being loaded into the First Chamber 510.
The sample processing device may further comprise a flow control device 330 as described above for controlling the flow of rinsing solution entering the first chamber. A flow control device 330B, which may be similar to flow control device 330, may also be used to control flow of the dissociation solution injected into the first chamber. In some embodiments, the dissociation solution is loaded in a syringe before being injected into the first chamber.
It is appreciated that the sample processing device disclosed herein may have different variations and combinations. For example, as shown in
Another embodiment of the sample processing device of the present disclosure is shown schematically in
Chamber 1, Port 1 and Port 2 may be an embodiment of the flow control device shown schematically in
Port 1 and Port 2 may comprise check valves, also known as one way valves that only allow fluids to enter and exit Chamber 1, respectively. The action of measuring and dispensing a set volume of fluid becomes very simple: decompressing Chamber 1 to allow fluids to enter via Port 1 and compressing Chamber 1 to push the fluids out via Port 2.
Alternatively, Chamber 1 may be a storage chamber which provides a pre-packaged solution that is needed for sample processing. For example, a lactated Ringer's solution, a balanced salt solution, a saline solution, a dissociation solution, a wash solution, a rinsing solution, a solution containing ethylenediaminetetraacetic acid (EDTA), and/or an enzyme solution, may be packaged in Chamber 1 as part of the device.
In another embodiment, the measuring chamber may comprise a syringe including a plunger, which may draw and/or dispense fluids of a pre-defined volume by pulling and pushing the plunger. In yet another embodiment, the measuring chamber may comprise a flexible tubing mounted on a peristaltic pump where fluid flows are controlled using the peristaltic pump.
Chamber 2 may be an embodiment of the first chamber 510 shown schematically in
After washing, a dissociation solution may be added to Chamber 2 to dissociate the tissue sample and release the cells. The temperature of the chamber may be set at a certain optimized temperature using a heating, cooling, and/or temperature control system to facilitate sample dissociation. For example, the device may be placed in an incubator, a water bath, and/or in contact with a constant temperature plate to hold the temperature at about 37 degrees Celsius for optimum enzyme digestion. The dissociation solution may comprise one or more enzymes to break down connective matrices, extracellular matrices, etc. For example, collagenase may be used at 37 degrees Celsius to break down collagen fibers. Other reagents, including proteinase, protease, trypsin, proteinase K, lyase, enzymes, lysis solutions, hyaluronidase, lipase, trypsin, liberase, DNase, deoxyribonuclease, pepsin, or mixtures thereof may also be used for tissue digestion. Outlets (Connector 1 and/or Connector 2) at the dissociation chamber (Chamber 2) may be closed off during digestion. Chamber 2 may be massaged, squeezed gently, rocked, shaken, partially squeezed and released back and forth, or otherwise agitated to facilitate mixing and promote efficient tissue dissociation. When the digestion act is completed, Connector 2 may be opened to allow the released cells to exit the dissociation chamber. At least one mesh in Chamber 2 may serve to remove or retain large pieces of debris. One or more chase wash acts comprising adding a wash solution may be applied to rinse off potentially trapped cells in Chamber 2 after digestion.
The sample processing device shown in
It is appreciated that the present disclosure is not limited to the specific configuration of the embodiment shown in
Another embodiment of a sample processing device of the present disclosure illustrated generally at 600 in
Any one or more of the mesh configurations illustrated in
Embodiments of sample processing devices disclosed herein may further comprise other parts, such as those illustrated in
Another embodiment of the present disclosure is a sample preparation device comprising a sample dissociation chamber (Chamber 1), a waste container (Chamber 2) and a cell refinement chamber (Chamber 3), illustrated generally at 700 in
Yet another embodiment of the present disclosure is a sample preparation device comprising two flexible sheets of materials, for example, plastic, bonded together in predefined patterns to form a sample dissociation chamber (Chamber 1), a waste container (Chamber 2) and a cell refinement chamber (Chamber 3), illustrated generally at 800 in
Another embodiment of the present disclosure comprises a downstream processing unit (DPU 1000 illustrated in
The downstream processing unit may comprise a microfluidic unit comprising at least one microfluidic device. The microfluidic device may comprise at least one channel dimension smaller than about 1 mm, for example, about 0.95 mm, about 800 μm, about 600 μm, about 500 μm, about 400 μm, about 300 μm, about 200 μm, about 150 μm, about 100 μm, about 80 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, and/or about 15 μm. The microfluidic device may also comprise channels of at least one substantially constant depth. For example, the microfluidic device may comprise channels having a depth of about 1 mm, about 800 μm, about 600 μm, about 500 μm, about 400 μm, about 300 μm, about 200 μm, about 150 μm, about 100 μm, about 80 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, or about 15 μm. The channel depths of the microfluidic device may be within 20% of a nominal channel depth. The microfluidic device may further comprise channels substantially on one surface, which may be substantially planar or curved. The microfluidic device may also comprise channels formed on one or more substantially planar surfaces.
The microfluidic device may be formed using microfabrication, nanofabrication, and/or micromachining techniques, including but not limited to photolithography, etching, reactive ion etching, deep reactive ion etching, wet etching, imprinting, injection molding, embossing, soft embossing, stereo lithography, molding, soft lithography, anodic bonding, ultrasound bonding, self assembling, and/or other fabrication techniques known in the art.
Embodiments of microfluidic units of the current disclosure may include devices disclosed in International Application PCT/US10/061866, International Publication WO 2011/079217 A1, U.S. Pat. No. 7,150,812 B2, U.S. Pat. No. 7,735,652, U.S. Pat. No. 8,021,614, U.S. Pat. No. 8,186,913 B2, United States Patent Application Publication No. US 2012/0063664 A1, United States Patent Application Publication No. US 2011/0294187 A1, which are herein incorporated by reference in their entireties for all purposes, devices employing Dean flows, inertial forces, centrifugal forces, deterministic lateral displacements, arrays of pillars, arrays of posts, pinch flows, magnetic structures, antibody components, cell capture moieties, protein capture moieties, deoxyribonucleic acid (DNA) moieties, ribonucleic acid (RNA) moieties, filtration, tangential flow filtration, ultrasound focusing, pinch flow, etc.
It is worth noting that some embodiments of the present disclosure, particularly those incorporating microfluidic devices disclosed in International Publication WO 2011/079217 A1, provide devices resistant to serious clogging and fouling, which until now have been a serious problem prohibiting the use of microfluidic devices for tangential flow filtration of digested fat tissue.
Another embodiment of the present disclosure comprises a hollow fiber unit to concentrate and/or wash the isolated cells.
In another embodiment of the downstream processing unit comprising a microfluidic device, the microfluidic device washes the cells and removes unwanted reagents. The downstream processing unit may comprise a buffer solution inlet to introduce a buffer solution to wash the cells. Cell wash may also be achieved using a microfluidic device designed to perform dialysis.
In yet another embodiment of the present disclosure, a downstream processing unit comprises a microfluidic device that reduces the enzyme concentration in the output of the downstream processing unit by a factor of greater than about 10, for example, the enzyme concentration is reduced by a factor of about 10, about 20, about 30, about 40, about 50, about 70, about 100, about 150, about 200, about 400, about 500, about 750, about 1,000, about 2,000, about 5,000, about 10,000, about 20,000, about 50,000, about 100,000, about 200,000, about 500,000, or about 1,000,000. One embodiment of the microfluidic device that may achieve such enzyme removal is disclosed in International Publication WO 2011/079217 A1, where the microfluidic device comprises pillars and employs at least one buffer stream, e.g. a stream of rinsing solutions, to wash cells.
In yet another embodiment of the present disclosure, a downstream processing unit comprises a microfluidic device that removes greater than 89% of the enzymes introduced during sample dissociation, for example, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.95%, about 99.98%, about 99.99%, about 99.995%, about 99.998%, about 99.999%, about 99.9995%, or about 99.9999% of the enzymes introduced during sample dissociation are removed.
In yet another embodiment of the present disclosure, a downstream processing unit comprises a microfluidic device that removes about 100% of the enzymes introduced during sample dissociation.
In yet another embodiment of the present disclosure, a downstream processing unit comprises a microfluidic device that provides an output having a collagenase concentration of less than about 0.1 mg/ml, for example, about 0.09 mg/ml, about 0.05 mg/ml, about 0.03 mg/ml, about 0.02 mg/ml, about 0.01 mg/ml, about 0.007 mg/ml, about 0.005 mg/ml, about 0.003 mg/ml, about 0.002 mg/ml, about 0.001 mg/ml, about 0.0005 mg/ml, about 0.0002 mg/ml, about 0.0001 mg/ml, about 0.00005 mg/ml, about 0.00002 mg/ml, about 0.00001 mg/ml, about 0.000001 mg/ml, or about 0.0000001 mg/ml.
In yet another embodiment of the present disclosure, a downstream processing unit comprises a microfluidic device that provides an output essentially free of enzymes introduced during sample dissociation. In yet another embodiment of the present disclosure, a downstream processing unit comprises a microfluidic device that provides an output free of enzymes introduced during sample dissociation.
In yet another embodiment of the present disclosure, a downstream processing unit comprises a microfluidic device that provides an output essentially free of collagenase introduced during sample dissociation. In yet another embodiment of the present disclosure, a downstream processing unit comprises a microfluidic device that provides an output free of collagenase introduced during sample dissociation.
Examples of microfluidic devices which may be used in any one or more of the sample processing devices disclosed herein are schematically illustrated in
Posts or pillars may be positioned in the microfluidic channels, for example, as illustrated in microfluidic channel 930 illustrated in
Microfluidic devices utilized for dialysis may be configured in series to form a cascade. An example is shown in
In some embodiments, the buffer used to form the buffer stream in a microfluidic device is a rinsing solution.
In yet another embodiment, the downstream processing unit comprises a dialysis membrane.
Yet another embodiment of the downstream processing unit 1000, schematically shown in
In another embodiment of the present disclosure, the downstream processing unit comprises multiple microfluidic devices to achieve the desired capacity, throughput, and functions for processing large volumes of output sample.
The transfer of fluids may be achieved using gravity, an external pressure, a vacuum, a positive pressure, a negative pressure, a head height, a pump, for example, a peristaltic pump, a mechanism configured to squeeze the bag, a roller that squeezes the bag, a plate that squeezes the bag, and/or other fluid transfer mechanisms known in the art. In one embodiment fluids may be transferred using a syringe. In another embodiment fluids may be transferred using an external air pressure applied to a chamber, for example Chamber 4 enclosing a bag (Chamber 3) containing cells, as shown in
In another embodiment of the present disclosure, the downstream processing unit comprises a cell culture chamber for culturing cells. The cell culture chamber may be connected using a connector, which allows the cell culture chamber to be detached. The cell culture chamber may be placed in an incubator, where the temperature and conditions for cell growth may be optimized, for example, at a temperature of about 37 degrees Celsius and about 5% carbon dioxide concentration. The cell culture chamber may further comprise materials permeable to air, for example, a filter membrane or silicone rubber film, allowing gas exchange during cell culture.
One or more chambers of sample processing devices as disclosed herein may comprise a mesh, multiple layers of mesh, and or a cascade of meshes (
Another embodiment of a sample processing device of the present disclosure comprises one or more chambers including two meshes, a second mesh in fluid communication downstream of a first mesh, where the pores of the second mesh are substantially smaller than the pores of the first mesh.
Yet another embodiment of a sample processing device of the present disclosure comprises one or more chambers including two membrane filters. A second of the membrane filters may be in fluid communication downstream of a first of the membrane filters. The pores of the second membrane filter may be substantially smaller than the pores of the first membrane filter.
Yet another embodiment of a sample processing device of the present disclosure comprises one or more chambers including a track-etched membrane filters.
Embodiments of sample processing devices or subcomponents thereof as disclosed herein may be constructed using materials including but not limited to thermoplastics, acrylonitrile butadiene styrene (ABS), acrylic (PMMA), celluloid, cellulose acetate, cyclic olefin copolymer (COC), cyclic olefin copolymer (COP), ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVOH), fluoroplastics (PTFE, alongside with FEP, PFA, CTFE, ECTFE, ETFE), ionomers, liquid crystal polymer (LCP), polyoxymethylene (POM or Acetal), polyacrylates (Acrylic), polyacrylonitrile (PAN or Acrylonitrile), polyamide (PA or Nylon), polyamide-imide (PAI), polyaryletherketone (PAEK or Ketone), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polycaprolactone (PCL), polychlorotrifluoroethylene (PCTFE), polyethylene terephthalate (PET), polycyclohexylene dimethylene terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoates (PHAs), polyketone, polyester, polyethylene (PE), polyetheretherketone (PEEK), polyetherketoneketone (PEKK), polyetherimide (PEI), polyethersulfone (PES), chlorinated polyethylene (CPE), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU), polytrimethylene terephthalate (PTT), polyurethane (PU), polyvinyl acetate (PVA), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), styrene-acrylonitrile (SAN), and/or acrylonitrile butadiene styrene (ABS). For medical applications, flexible plastic sheets such as polyvinyl chloride (PVC), polyurethane (PU), ethylene-vinyl acetate (EVA), polyamide (PA or Nylon) may be used as the sheet material. In some embodiments a sample processing device as disclosed herein may comprise two flexible sheets bonded together and defining one or more chambers therebetween. In other embodiments a sample processing device as disclosed herein may comprise a flexible sheet bonded to a rigid or semi-rigid material (for example, a thick sheet of plastic and/or any one or more of the materials disclosed above) and defining one or more chambers between the flexible sheet and the rigid or semi-rigid material.
The thickness of the sheet material may be, in some embodiments, between about 0.1 mm and about 0.8 mm, for example, about 0.1 mm, about 0.15 mm, about 0.2 mm, about 0.25 mm, about 0.3 mm, about 0.35 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, or about 0.8 mm. The thickness of the sheet material may be, in other embodiments, between about 0.2 mm and about 0.4 mm, for example, about 0.2 mm, about 0.25 mm, about 0.3 mm, about 0.35 mm, or about 0.4 mm.
Materials for membrane filters and/or meshes may include, but are not limited to cellulose acetate (CA), glass microfibre (GMF), polyethersulphone (PES), polypropylene (PP), regenerated cellulose (RC), polyamide (PA or Nylon), polytetrafluoroethylene (PTFE), and/or polyvinylidene fluoride (PVDF).
The thickness of the mesh material may be, in some embodiments, between about 10 μm and about 1,000 μm, for example, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 175 μm, about 200 μm, about 250 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm or about 1 mm. The thickness of the mesh material may be, in other embodiments, between about 50 μm and about 300 μm, for example, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 125 μm, about 140 μm, about 160 μm, about 180 μm, about 200 μm, about 220 μm, about 250 μm, about 275 μm, or about 300 μm.
Embodiments of the present disclosure may be constructed using standard plastic manufacturing techniques, including, but not limited to plastic welding, heat sealing, injection molding, embossing, glue bonding, ultraviolet light (UV) cured adhesive bonding, solvent bonding, hot gas welding, freehand welding, speed tip welding, extrusion welding, contact welding, hot plate welding, high frequency welding, radio frequency welding, injection welding, ultrasonic welding, friction welding, spin welding, laser welding, and/or solvent welding.
Embodiments of the present disclosure, for example, the sample processing device shown schematically in any of
In some embodiments of the present disclosure the sample preparation device is sterile. In some embodiments of the present disclosure the sample preparation device is single-use. Further, in some embodiments of the present disclosure the sample preparation device is a substantial protective barrier providing an isolated environment, where samples are protected from direct physical contact, or fluid contact, for example, through unfiltered air flow, with the external environment and/or operating personnel to minimize or avoid contamination and infection risks.
It is appreciated that embodiments of the present disclosure may serve as a protective barrier that substantially reduces or eliminates any direct physical contact, fluid connection, and/or air flow connection between a tissue sample and the ambient environment. Any part of embodiments of the devices disclosed herein that may be in direct physical contact, fluid connection, and/or unfiltered air flow connection with the sample may be sterile and/or single use. It is appreciated that embodiments of the devices disclosed herein may substantially protect a sample from contamination risks and operators from infection risks.
It is appreciated that the present disclosure enables the design of tissue processing devices that are very easy to use. It is also appreciated that present disclosure may drastically simplify the manufacturing process and reduce the manufacturing cost of such tissue processing devices. It is further appreciated that the embodiments of the present disclosure enables a tissue sample to be processed safely substantially free of contamination and infection risks using a device that substantially isolates the sample from the surrounding laboratory or hospital environment.
Human lipoaspirate was processed using a method comprising the acts depicted in
Initially, Clamps 1 and 2 were applied to close off Connectors 1 and 2 (
The tissue dissociation act started with closing Clamps 1 and 2 and adding a dissociation solution into Chamber 1 from the Y insertion site (
After dissociation, Clamp 1 was open to allow released cells to enter a debris removal chamber (Chamber 4 in
The solution coming out of Chamber 4 was then fed under gravity into a microfluidic device 1100, illustrated in
The outputs of the microfluidic device are shown in
Cells isolated using the disclosed method and device were further characterized for total nucleated cell count and adherent viable nucleated cell count using an ADAM MC automated mammalian cell counter. The output of Chamber 4 had about 6.0×105 total nucleated cells per ml of lipoaspirate processed. Alpha-MEM supplemented with 10% Fetal Bovine Serum (FBS) was used as the culture medium for adherent viable nucleated cell count. The output of Chamber 4 was sampled and cultured at 37 degrees Celsius for 3 days in the culture medium in cell culture chambers, for example, a cell culture dish or a cell culture flask. After 3 days, the culture medium was discarded and the cell culture chambers were washed using a Dulbecco phosphate buffered saline solution. Cells adhering to the culture chamber were then released from the chamber surface using Trypsin for 3 minutes. The adherent viable nucleated cell count was about 1.5×105 per ml of lipoaspirate processed.
Human lipoaspirate was processed using a sample processing bag device as depicted in
Human lipoaspirate was collected from a consented donor using tumescent liposuction and processed within 6 hours from collection. The sample was shipped and stored at 4 degrees Celsius before processing.
Initially, Stopcock 1 was set to a position that connects Chamber 1 and Chamber 2. A dissociation solution comprising 100 mg of collagenase, 100 mg of hyaluronidase, and 20,000 U of deoxyribonuclease was loaded into Syringe 2. The sample processing bag device was connected to a bag of rinsing solution comprising lactated Ringer's injection solution using the Spike.
About 75 ml of the lipoaspirate sample was injected into Chamber 1 from Port 1 using a syringe with a catheter tip.
A wash act was applied to clean the lipoaspirate sample. To begin a wash cycle, Stopcock 1 was set to a closed position to disconnect Chamber 1 fluidicly from chambers 2 and 3. About 100 ml of lactated Ringer's injection solution was injected into Chamber 1 using Syringe 1 and Stopcock 2 as a flow control device, which worked using the following sequence of acts: (a) switch Stopcock 2 to connect the rinsing solution to Syringe 1; (b) draw the rinsing solution into Syringe 1; (c) switch Stopcock 2 to connect Syringe 1 to Chamber 1; (d) inject the rinsing solution from Syringe 1 into Chamber 1. The sequence may be repeated until the desired volume of solutions is added to the dissociation chamber.
Chamber 1 was then massaged to mix the rinsing solution with the sample, and Stopcock 1 was switched to drain excess fluids, i.e. the waste solution, into Chamber 2. After draining, the first wash cycle was complete. The wash cycle was repeated twice.
The wash act may comprise one or many wash cycles, for example, one, two, three, four, or five cycles.
After washing, the dissociation solution was added to Chamber 1. About 100 ml of the rinsing solution was also added to Chamber 1. The sample processing bag device was then incubated at 37 degrees Celsius for an incubation time of about 60 minutes. Chamber 1 was massaged during this time to mix the sample with the dissociation solution. In some embodiments, other incubation times may also be used, for example, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 75 minutes, about 90 minutes, or about 120 minutes.
After incubation, individual cells released from the sample were passed through Stopcock 1 into Chamber 3, while large tissue debris were captured by Mesh 1 and left in Chamber 1. Mesh 2 in Chamber 3 further removed large adipocytes and debris from the dissociated sample. The non-fat cells, including pericytes, adipose derived stem cells and progenitor cells, were then collected at the Outlet Port of Chamber 3.
To concentrate the released cells and removed residual erythrocytes and debris, the released cells collected at the Outlet Port of Chamber 3 of the sample processing bag device were then run through a first microfluidic device disclosed in International Publication WO 2011/079217 A1. The microfluidic device comprised 73 modules arranged on a surface of a substrate. Each module comprised about 1,300 pillars configured into four rows. The microfluidic device comprised channels of substantially the same depth, between about 35 μm and about 50 μm. In another embodiment, the microfluidic device may comprise channels having a depth of between about 30 μm and about 80 μm, for example, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm, or about 80 μm. The cells at the output of the microfluidic device were concentrated by a factor of about 3. In another embodiment of the present invention, a microfluidic device may be used to concentrate the cells by a factor of greater than about 2.5, for example, by a factor of about 3, about 4, about 5, about 6, about 8, about 10, about 12, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 80, about 100, or about 125.
To remove enzymes, the cells were processed through a second microfluidic device disclosed in International Publication WO 2011/079217 A1. The second microfluidic device comprised 83 modules arranged on a surface of a substrate. Each module comprised about 900 pillars configured into four rows. A stream of the rinsing solution was introduced in each module and the cells were transferred into the rinsing solution stream, separated from the enzymes.
An enzyme-linked immunosorbent assay (ELISA) was applied to measure the residual amount of collagenase after the second microfluidic device. The result shows that the collagenase concentration was reduced by a factor of 1,000, and the refined cells contain less than 0.001 mg/ml of collagenase.
Having thus described several aspects of at least one embodiment, it is to be appreciated various alterations, modifications, combinations and improvements will readily occur to those skilled in the art. Such alterations, modifications, combinations and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the disclosure. Accordingly, the foregoing description and drawings are by way of example only.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US12/68233 | 12/6/2012 | WO | 00 | 6/5/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61567920 | Dec 2011 | US |
