JP2015500031A - Method and apparatus for separating non-adipocytes from adipose tissue - Google Patents

Abstract

According to one aspect of the present disclosure, an apparatus for separating non-adipocytes from an adipose tissue sample is provided. The apparatus comprises a first sheet material, a second sheet material bonded to the first sheet material, and a plurality of chambers defined between the first sheet material and the second sheet material. 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 purification including an inlet and outlet in fluid communication with the sample dissociation chamber A plurality of chambers including a chamber. [Selection] Figure 13A

Description

  Many techniques in biology and medicine, such as cell sorting, flow cytometry, cell assays, and cell therapy, are based on dissociating tissues and separating individual cells. The processing of tissue samples often involves multiple operations (eg, fragmentation, washing, enzyme digestion, dissociation, culture, mixing, aggregation, residue removal, and concentration). In the laboratory, these operations are often performed manually with the sample being manually transferred from test tube to test tube in an open environment. Such manual processes require highly trained personnel, and handling biological samples creates a potential risk of contamination and infection.

  In recent years, research and medical tissues have become increasingly interested in separating cells from adipose tissue. Several techniques have been developed to safely remove a portion of adipose tissue from a patient or animal. For example, the tumecent liposuction technique and the water jet liposuction technique are widely used to remove adipose tissue from a patient. Adipose tissue includes fat cells that contain fat and other non-adipocytes that hold the tissue. The constituent cells of adipose tissue, their roles and interactions are not fully understood, and academic and clinical research on this has been actively conducted.

  In studying adipose tissue, it may be desirable to dissociate the tissue and isolate the constituent cells. The process may involve release of constituent cells, removal of debris and unwanted cells, enrichment and enrichment of subject cells, and cell washing. Such a process is difficult and may require laboratories with highly trained operators, expensive equipment setups, and appropriate biosafety measures. By performing a plurality of operations, the target cells are significantly lost, and it may be difficult to separate rare and rare cells, resulting in low reliability. Furthermore, when human samples are used, the risk of cross-contamination and infection can be substantial.

  Therefore, having a method for effectively separating target cells (particularly cells other than fat cells) from adipose tissue and having a device that makes it easy and safe to separate non-adipocytes from adipose tissue desired.

  Bags containing flexible plastic sheets are widely used to collect, process and store biological tissue samples (eg peripheral blood, umbilical cord blood, blood components, plasma, bone marrow, aspirated adipose tissue, etc.) It has been. The advantage of the bag is that it is flexible and expandable, and because it has the ability to change the internal volume, it can accommodate samples of different volumes. To assist in sample processing, a number of individual bags are often fluidly connected using external tubes, thereby forming a system. Such bag and tube systems are widely used in sample processing (eg, blood sorting, cell separation, etc.). On the other hand, if a larger number of processing operations are integrated, the bag and tube system will soon become longer and more complex, making it difficult to use and difficult to manufacture. Such systems tend to be spaghetti and tangled with many components hanging from each other. In using such a device, an operator needs advanced training and long-term practical training in order to set up a complicated device. The operator also needs to be more careful to install the various parts in the right place in the right order. In addition, these devices and systems can often be difficult and expensive to manufacture because many bags, components, and lengths of tubing need to be assembled after they have been made individually. The assembly of such a device is labor intensive and can result in leakage and contamination risks, i.e. system failure. For clinical applications, these devices are often single use, and their reliability is important. Significant labor and device failure risks are major obstacles to these conventional spaghetti-like systems.

  According to one aspect of the present disclosure, an apparatus for separating non-adipocytes from an adipose tissue sample is provided. The apparatus comprises a first sheet material, a second sheet material bonded to the first sheet material, and a plurality of chambers defined between the first sheet material and the second sheet material. 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 purification including an inlet and outlet in fluid communication with the sample dissociation chamber A plurality of chambers including a chamber.

  According to some embodiments, the sample dissociation chamber further comprises a mesh filter including pores having pore sizes in the range of 70 μm to 300 μm.

  According to some embodiments, the apparatus further includes a mesh filter that is included in the cell purification chamber and includes pores having a pore size ranging from 20 μm to 50 μm.

  According to some embodiments, the sample dissociation chamber further includes a first mesh filter that includes pores having a first pore size. The cell purification chamber further includes a second mesh filter including pores having a second pore size smaller than the first pore size.

  According to some embodiments, the apparatus further includes means for controlling the fluid connection between the sample dissociation chamber, the waste collection chamber, and the cell purification chamber.

  According to some embodiments, the means for controlling the fluid connection includes a stopcock.

  According to some embodiments, the apparatus is a flow control device configured to introduce at least one of a wash solution and a dissociation solution into the sample dissociation chamber, the flow control device being in fluid communication with the sample dissociation chamber. Further included is a flow control device having an outlet.

  According to some embodiments, the apparatus further includes means for applying pressure to one of the sample dissociation chamber and the cell purification chamber.

  According to some embodiments, the device is a downstream processing device that is in fluid communication with an outlet of the cell purification chamber, wherein the fluid output from the cell purification chamber is transferred to one or more target cells. A downstream comprising at least one microfluidic device configured to sort into a first solution having a concentration and a second solution having a concentration less than the concentration of the first solution of one or more target cells; A processing device is further included. The target cells include non-adipocytes separated from the adipose tissue sample.

  According to one aspect of the present disclosure, a sterilized and substantially separated adipose tissue processing system is provided. The system includes an inlet, an outlet, and a tissue processing chamber including at least one mesh filter disposed between the inlet of the tissue processing chamber and the outlet of the tissue processing chamber, and the same housing as the tissue processing chamber A waste collection chamber included in a tissue processing chamber, including an inlet that is in fluid communication with an outlet of the tissue processing chamber, a residue removal chamber that includes a residue removal mechanism, and a tissue processing chamber A waste collection chamber comprising one of the sample collection chambers in fluid communication with the waste collection chamber.

  According to one aspect of the present disclosure, a method for processing an adipose tissue sample in a tissue processing system is provided. The method includes introducing an adipose tissue sample to be processed into a first chamber through an inflow port of the first chamber, processing the adipose tissue sample in the first chamber, and treating cells in the first chamber. Transporting from the first chamber through the outlet to the second chamber contained in the same housing as the first chamber through the inlet of the second chamber.

  According to some embodiments, processing the adipose tissue sample includes dissociating the adipose tissue sample.

  According to some embodiments, processing the adipose tissue sample includes removing excess fluid from the adipose tissue sample in the first chamber.

  According to some embodiments, processing the adipose tissue sample includes rinsing the adipose tissue sample in the first chamber with a wash solution.

  According to some embodiments, processing the adipose tissue sample comprises washing the adipose tissue sample in the first chamber using a wash solution and in the first chamber using a dissociation solution comprising at least one enzyme. Dissociating the adipose tissue sample.

  According to some embodiments, the dissociation solution comprises collagenase.

  According to some embodiments, the dissociation solution comprises collagenase, deoxyribonuclease, and hyaluronidase.

  According to some embodiments, dissociating the adipose tissue sample with the dissociation solution is performed at about 37 degrees Celsius.

  According to some embodiments, the method further includes removing the residue using a mesh filter included in the second chamber.

  According to some embodiments, the mesh filter has a pore size ranging from 15 micrometers to 100 micrometers.

  According to some embodiments, the method retains the sample in the first chamber and passes the waste fluid through the mesh filter contained in the first chamber and the first outlet of the first chamber with the first chamber. It further includes transporting to a third chamber included in the same housing through an inlet of the third chamber.

  According to some embodiments, the method further comprises enriching for the non-adipocyte population using a microfluidic device.

  According to some embodiments, the non-adipocyte is a stem cell.

  According to some embodiments, the method further comprises collecting cells from the tissue processing system.

  According to some embodiments, the method is a downstream processing device that fluidly communicates cells to an outlet of the second chamber, the cells comprising a first solution having a first concentration of non-adipocytes; And further processing in a downstream processing device comprising at least one microfluidic device configured to sort into a second solution having a concentration less than the concentration of the first solution of non-adipocytes.

  According to some embodiments, the harvested cells are stromal vascular cell group cells.

  According to one aspect of the present disclosure, an apparatus for separating cells from adipose tissue is provided. The apparatus comprises a first sheet material, a second sheet material bonded to the first sheet material, and a plurality of chambers defined between the first sheet material and the second sheet material. 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 tissue dissociation chamber, and a cell purification including an inlet and outlet in fluid communication with the sample dissociation chamber A plurality of chambers including a chamber.

  According to some embodiments, the sample dissociation chamber further comprises a filtration mesh.

  According to some embodiments, the apparatus further includes a filtration mesh included in the cell purification chamber.

  According to some embodiments, the sample dissociation chamber further includes a first filtration mesh that includes pores having a first pore size. The cell purification chamber further includes a second filtration mesh that includes pores having a second pore size.

  According to some embodiments, the second pore size is smaller than the first pore size.

  According to some embodiments, the apparatus further includes means for controlling the fluid connection between the sample dissociation chamber, the waste collection chamber, and the cell purification chamber.

  According to some embodiments, the means for controlling the fluid connection includes a stopcock.

  According to some embodiments, the apparatus is a flow control device configured to introduce one of a wash solution and a dissociation solution into the sample dissociation chamber, wherein the outlet is in fluid communication with the tissue dissociation chamber. And a flow control device.

  According to some embodiments, the apparatus further includes means for applying pressure to one of the sample dissociation chamber and the cell purification chamber. This means may be arranged between the material of the first sheet and the material of the second sheet. This means may be arranged in the vicinity of the first sheet material and / or the second sheet material.

  According to some embodiments, the device is a downstream processing device that is in fluid communication with an outlet of the cell purification chamber, wherein the fluid output from the cell purification chamber is transferred to one or more target cells. A downstream comprising at least one microfluidic device configured to sort into a first solution having a concentration and a second solution having a concentration less than the concentration of the first solution of one or more target cells; A processing device is further included.

  According to one aspect of the present disclosure, a substantially separated adipose tissue processing system is provided. The system includes an inlet, a first outlet, a second outlet, and at least one filtration mesh disposed between the inlet of the adipose tissue processing chamber and the first outlet of the adipose tissue processing chamber. Residue removal including a tissue processing chamber, a waste collection chamber contained in the same housing as the fat tissue processing chamber, wherein the inlet is in fluid communication with the first outlet of the fat tissue processing chamber, and a residue removing mechanism And a waste collection chamber including one of the sample collection chambers contained in the same housing as the adipose tissue processing chamber and in fluid communication with the second outlet of the adipose tissue processing chamber.

  According to some embodiments, the system includes a fluid volumetric chamber disposed in the same housing as the adipose tissue processing chamber, wherein the inlet is in fluid communication with the inlet of the adipose tissue processing chamber. And a fluid volume measuring chamber.

  According to some embodiments, each of the fluid volumetric chamber inlet and the fluid volumetric chamber outlet includes a check valve.

  According to some embodiments, the first outlet and the second outlet include a stopcock outlet in fluid communication with the adipose tissue processing chamber.

  According to one aspect of the present disclosure, a method for separating cells from adipose tissue is provided. The method includes introducing a sample to be processed into the tissue processing chamber through an inlet port of the tissue processing chamber, processing the sample in the tissue processing chamber, and passing cells through the outlet of the tissue processing chamber. And discharging into the sample storage chamber through an inlet of the sample storage chamber contained in the same housing as the tissue processing chamber.

  According to some embodiments, the method retains a cell sample in a tissue processing chamber and passes waste fluid through a mesh filter included in the tissue processing chamber and a first outlet of the tissue processing chamber. And transporting through a waste collection chamber inlet to a waste collection chamber contained in the same housing.

  According to some embodiments, the method further includes extracting a cell sample from the tissue processing system.

  According to some embodiments, the method is a downstream processing device that fluidly communicates an extracted cell sample to an outlet of a sample storage chamber, wherein the extracted cell sample is applied to one or more subjects. At least one microfluidic device configured to sort into a first solution having a first concentration of cells and a second solution having a concentration lower than the concentration of the first solution of one or more target cells; It further includes processing in a downstream processing apparatus.

  According to some embodiments, processing the tissue sample in the tissue processing chamber includes introducing a tissue wash solution into the tissue processing chamber.

  According to some embodiments, processing the tissue sample in the tissue processing chamber further includes introducing a tissue dissociation solution into the tissue processing chamber.

  According to one aspect of the present disclosure, a method for separating cells from adipose tissue is provided. The method introduces a suctioned adipose tissue sample to be processed into a chamber containing a mesh, removes excess fluid from the sample, and removes at least one dissociated sample to remove adipocytes and debris. Passing through one mesh and substantially removing red blood cells from the sample and concentrating the sample at least three times using a microfluidic device.

  According to some embodiments, the method further includes washing the sample with a wash solution.

  According to some embodiments, the method further comprises mixing and culturing the sample and a dissociation solution comprising at least one enzyme.

  According to some embodiments, the microfluidic device includes a plurality of microfluidic channels having a substantially constant depth and having at least one dimension that is less than 750 micrometers. Still, at least two microfluidic channels are disposed on one surface of the substrate.

  According to some embodiments, the dissociation solution comprises collagenase and the sample and dissociation solution are cultured in the range of about 20 degrees Celsius to about 40 degrees Celsius.

  According to some embodiments, the dissociation solution comprises collagenase and deoxyribonuclease, and the sample and dissociation solution are incubated at about 37 degrees Celsius.

  According to some embodiments, the microfluidic device further includes struts configured to sort the sample into a non-adipose nucleated cell fraction.

  According to some embodiments, the microfluidic device is configured to wash non-adipocytes and use a washing solution to substantially remove the enzyme in the dissociation solution.

  According to one aspect of the present disclosure, a substantially separated adipose tissue processing system is provided. The system includes a sample wash and dissociation chamber including three inflow ports, a first outflow port and a second outflow port, and a mesh disposed between the three inflow ports and the first outflow port and the second outflow port. An agglomerate reduction chamber comprising: an inlet connector fluidly connecting to a 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 removal of dissociated cells and further residues having an inlet in fluid communication with the outlet, a waste solution collection chamber having an inlet in fluid communication with the second outlet port of the sample washing and dissociation chamber, and a micro A fluidic device.

  According to some embodiments, the microfluidic device includes a plurality of microfluidic channels having a substantially constant depth and at least one dimension that is less than 750 micrometers.

  According to some embodiments, at least two of the plurality of microfluidic channels are arranged on one surface of the substrate.

  According to some embodiments, each of the sample wash / dissociation chamber, the agglomerate reduction chamber, the reservoir, and the waste solution collection chamber are contained within the same sealed package.

  The accompanying drawings are not intended to be drawn to scale. In these drawings, identical or nearly identical components illustrated in the various drawings are represented by like numerals. For simplicity, not all components are labeled in each drawing. All drawings are to be considered schematic unless otherwise specified.

3 is a flowchart of one method according to one embodiment of the present disclosure. 3 is a flowchart of one method according to one embodiment of the present disclosure. 1 is a schematic diagram of a sample processing apparatus according to one embodiment of the present disclosure. 1 is a schematic diagram of a flow control device according to one embodiment of the present disclosure. FIG. 1 is a schematic diagram of a flow control device according to one embodiment of the present disclosure. FIG. 1 is a schematic diagram of a sample processing apparatus according to one embodiment of the present disclosure. 1 is a schematic diagram of a sample processing apparatus according to one embodiment of the present disclosure. 1 is a schematic diagram of a sample processing apparatus according to one embodiment of the present disclosure. 1 is a schematic diagram of a sample processing apparatus according to one embodiment of the present disclosure. 1 is an elevation view of a sample processing apparatus according to one embodiment of the present disclosure. FIG. FIG. 3B is an isometric view of the sample processing apparatus shown in FIG. 3A. FIG. 3C is an exploded view of a portion of the chamber of the apparatus shown in FIG. 3A. 3B is an exploded view of a portion of the chamber of the apparatus shown in FIG. 3A. FIG. It is sectional drawing which looked at one part of the chamber of the apparatus shown to FIG. 3A from the side. 3B is an elevation view of the sample processing apparatus shown in FIG. 3A including a clamp as desired. FIG. 1 is an elevation view of a sample processing apparatus according to one embodiment of the present disclosure. FIG. 1 is an elevational view of one chamber of a sample processing apparatus according to one embodiment of the present disclosure. FIG. It is sectional drawing which looked at one part of the chamber shown to FIG. 5A from the side. 1 is an elevational view of one chamber of a sample processing apparatus according to one embodiment of the present disclosure. FIG. It is sectional drawing which looked at one part of the chamber shown to FIG. 6A from the side. It is sectional drawing which looked at one chamber of the sample processing apparatus which concerns on one Embodiment of this indication from the side. 1 is an elevational view of one chamber of a sample processing apparatus according to one embodiment of the present disclosure. FIG. FIG. 8B is an exploded view of the chamber shown in FIG. 8A. 1 is an elevational view of one chamber of a sample processing apparatus according to one embodiment of the present disclosure. FIG. FIG. 9B is an exploded view of the chamber shown in FIG. 9A. 1 is an elevation view of a portion of a sample processing apparatus according to one embodiment of the present disclosure. FIG. FIG. 10B is an exploded view of a portion of the sample processing apparatus shown in FIG. 10A. 1 is an elevation view of a sample processing apparatus according to one embodiment of the present disclosure. FIG. 1 is an elevation view of a sample processing apparatus according to one embodiment of the present disclosure. FIG. 1 is an elevational view of a sample processing apparatus according to one embodiment of the present disclosure. FIG. FIG. 13B is a cross-sectional view of a portion of the chamber of the apparatus shown in FIG. 13A. FIG. 13B is a cross-sectional view of a portion of the chamber of the apparatus shown in FIG. 13A. 1 is an elevation view of a sample processing apparatus according to one embodiment of the present disclosure. FIG. FIG. 3 illustrates a portion of a microfluidic device included in some embodiments of the present disclosure. FIG. 3 illustrates a microfluidic device included in some embodiments of the present disclosure. FIG. 3 illustrates a microfluidic device included in some embodiments of the present disclosure. FIG. 3 illustrates a microfluidic device included in some embodiments of the present disclosure. FIG. 3 illustrates a microfluidic device included in some embodiments of the present disclosure. 1 is an elevation view of a sample processing apparatus according to one embodiment of the present disclosure. FIG. FIG. 3 illustrates a microfluidic device included in some embodiments of the present disclosure. 2 is a photograph of a fluid processed in one embodiment of the present disclosure. 2 is a photograph of a fluid processed in one embodiment of the present disclosure.

  This disclosure is not limited in its application to the details of construction and construction of the components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or carried out in various ways. Also, the terminology and terms used herein are used for explanatory purposes and should not be construed in a limiting sense. “Including”, “comprising”, “having”, “containing”, “with”, and variations thereof herein are the items preceding these terms and their equivalents In addition, it is intended to include additional items.

  As used herein, the terms “sample”, “tissue sample”, “adipose sample”, and “adipose tissue” may include adipose tissue samples (eg, aspirated adipose tissue), which terms are interchangeable Used for.

  As used herein, the term “microfluidic device” refers to at least one fluid channel (which may be substantially flat or curved) formed on a substantially single surface; May refer to a device having at least one lateral channel dimension that is less than about 1 mm. The lateral channel dimension can be, for example, the width or depth of the channel.

  It has been found desirable to have a method for effectively separating target cells from adipose tissue and to have a device that makes it easy and safe to separate cells from adipose tissue. For research and clinical applications, it has been found desirable to facilitate multiple operations to process adipose tissue samples so that human errors are minimized. Furthermore, it has been found important that the adipose tissue sample is processed in a substantially “separated” environment. In such an environment, a barrier is provided to separate the fat sample from direct physical contact with the external environment and / or operating personnel or fluid contact, for example through an unfiltered air stream, thereby preventing contamination and infection. Risk is minimized or avoided. It is also desirable to have a system and apparatus for adipose tissue processing in which multiple components and compartments are integrated as one piece, thereby providing a substantially isolated environment for the sample. Has been issued. The system and apparatus for adipose tissue processing is preferably easy to use and manufacture and has a low risk of failure. For many clinical and research applications, it may be preferred that any part of such devices and systems that come in direct contact with the adipose tissue sample is sterilized and disposable.

  Aspects and embodiments of the present disclosure provide methods for separating specific constituent cell populations from adipose tissue samples. Other aspects and embodiments of the present disclosure are for enabling a method for separating specific constituent cell populations from adipose tissue samples in an integrated, smoothed, safe and easy-to-use manner. Providing equipment.

  Aspects and embodiments of the present disclosure provide an integrated device that includes multiple compartments for processing adipose tissue samples. Such multiple compartments include, for example, sample collection, washing, stratification, mixing, heating, cooling, filtration, digestion, storage, fluid transport and handling, cell labeling, sample processing, dissociation, waste fluid collection, clump removal, Includes but is not limited to compartments configured and arranged for residue removal, cell enrichment, cell enrichment, cell separation, cell culture, growth, culture, differentiation, expansion, etc. An integrated device according to embodiments of the present disclosure may also include a valve for controlling fluid flow between the compartments, for example. Such an apparatus may be useful for the integration and rationalization of multiple operations of adipose tissue sample processing (eg, separation of cells from adipose tissue), and multiple functions (eg, enzymatic digestion, adipose tissue dissociation, washing) Waste liquid collection, residue removal, cell concentration, labeling with antibodies, labeling with magnetic beads, cell expansion, etc.). Such devices can be particularly useful in applications where safety, easy use, and easy manufacture are important. Some aspects and embodiments of the present disclosure include a method for using such an apparatus.

  One embodiment of the disclosed method for separating cells from adipose tissue is to dissociate adipose tissue, release constituent cells, collect released cells, and remove tissue debris. Including, but not limited to. The method may further include an adipose tissue cleaning operation prior to tissue dissociation. The adipose tissue cleaning operation can include removing or draining unwanted or excess fluid from the adipose tissue sample. Such unwanted fluids can include blood, bodily fluids, and tubular solutions. The adipose tissue cleaning operation may further comprise washing or washing the adipose tissue with a washing solution. The method can further include one or more operations to enrich or purify the released cells. In addition, the method may further include collecting waste fluid from the process. Other embodiments of the disclosed method for separating cells from adipose tissue include removing excess fluid from the adipose tissue, dissociating the adipose tissue and releasing constituent cells, and removing unwanted cells and debris. Including, but not limited to. The method may further include one or more of washing the adipose tissue sample, enriching the subject cells, washing the subject cells, and performing immunoisolation, eg, using an antibody. In one embodiment, enriching and / or washing the subject cells can be performed using at least one microfluidic device. In other embodiments, one or more operations may use centrifugation. In still other embodiments, one or more operations can be performed utilizing hollow fibers.

  In one embodiment of the present disclosure, a method for separating a non-adipocyte population from adipose tissue is provided. This method involves removing excess fluid from adipose tissue, washing adipose tissue with a buffer, eg dissociating adipose tissue with ultrasound or a dissociation solution containing enzymes, adipocytes, free oil (Free oil), removal of matrix fibers, and tissue debris, reduction of red blood cells, and enrichment of subject cells, but are not limited to these. Cell enrichment operations may be achievable using centrifuges, filters, or microfluidic devices. The method may further include one or more of lymphocyte reduction, cell washing, and immune sequestration. Removing excess fluid from adipose tissue, washing adipose tissue with buffer, dissociating adipose tissue, removing adipocytes, free oil, matrix fibers, and tissue debris, and cell washing Can be performed using strainers including, for example, subsidence under gravity, centrifugation, and / or mesh filters.

  A flowchart of one embodiment of a method for separating a non-adipocyte population from adipose tissue is indicated generally by the reference numeral 100 in FIG. During the performance of liposuction procedures, a tumenic fluid is often introduced into a patient to minimize blood loss, to harden tissue, and to provide local anesthesia. The tumecent solution may contain lidocaine at a concentration of 0.05% and epinephrine at a concentration of 1: 1,000,000. The method includes an operation (operation 110) of removing excess fluid from the aspirated adipose tissue. Excess fluid may include blood and often tumenic fluid. It should be noted that the tumescent solution can interfere with downstream processing operations and target cell separation. In one embodiment, the removed excess fluid sample may be stratified into an adipose tissue layer and an excess fluid layer using gravity settlement or centrifugation. This is because adipose tissue is less dense than excess fluid. The adipose tissue layer and excess fluid can then be separated into different containers, whereby the adipose tissue and excess fluid can be separated. For example, a wash solution containing saline, lactated Ringer's solution, Hank's balanced salt solution, or phosphate buffered saline can be added to the adipose tissue to wash the adipose tissue, and the stratification process more thoroughly cleans the adipose tissue And can be repeated to remove excess fluid. In other embodiments, excess fluid may be drained using a strainer that includes a mesh. The washing solution can be added to the adipose tissue and drained using a strainer to wash the tissue. This washing process may be repeated. In some embodiments, the strainer ranges from about 30 micrometers (μm) to about 1 millimeter (mm) (eg, about 30 μm, about 50 μm, about 70 μm, about 85 μm, about 100 μm, about 120 μm, about 140 μm, about 200 μm, about 300 μm, about 500 μm, about 700 μm, or about 1 mm). In other embodiments, the strainer may include pores having pore sizes in the range of about 70 μm to about 500 μm (eg, about 70 μm, about 100 μm, about 140 μm, about 200 μm, about 300 μm, or about 500 μm). More particularly, the strainer may include a mesh filter having a pore size in the range of about 70 μm to about 200 μm (eg, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm, or about 200 μm). In other embodiments of the present disclosure, the strainer has a smaller pore size than the adipose tissue so that the adipose tissue is retained by the strainer. In order to effectively remove excess fluid, the washing operation including adding the washing solution and removing the washing solution is about 1 to about 10 times (e.g. once, twice, three times, four times). 5 times, 6 times, 8 times, or 10 times). The ratio of the volume of fat sample to the volume of wash solution added with each wash ranges from about 1: 0.2 to about 1:10 (eg, about 1: 0.2, about 1: 0.3, about 1). : 05, about 1: 0.7, about 1: 1, about 1: 2, about 1: 3, about 1: 5, or about 1:10). For example, 100 ml of aspirated adipose tissue collected using Tumecent liposuction can be mixed with 100 ml of lactated Ringer's solution and drained using a nylon mesh having pores with a pore size of about 140 μm. This process can be performed three times to completely remove excess fluid. In other embodiments, each washing operation includes adding a washing solution to the sample and removing the washing solution from the sample. The washing solution has a range of 0.6 to 4 times the volume of the sample (for example, 0.6, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.5 of the sample volume). 3 or 4 times) and the washing operation can be performed once, twice, three times or four times. In other embodiments, the excess fluid removal operation can be combined with a stratification operation using gravity prior to discharge using a strainer. In yet other embodiments, prior to stratification or draining of fluid using a mesh strainer, a cleaning solution may be added and mixed to the raw adipose tissue, thereby diluting excess fluid. By adding a cleaning solution, stratification or draining can be more effective.

  In other embodiments of the present disclosure, the excess fluid removal operation may be performed by placing 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 include a means for fluid control (eg, a pinch valve). To remove excess fluid, excess fluid can be drained through the outlet. As the sample approaches the outlet, the outlet can be closed using fluid control means. The outlet may have a smaller dimension than the adipose tissue sample or a larger dimension to allow passage of the adipose tissue sample. This operation may be performed manually or may be performed with the aid of a sensor that detects the sample relative to the outlet (such as an optical sensor or an infrared sensor). To wash or wash the adipose tissue sample, a wash solution is added to the adipose tissue sample, and the excess fluid removal operation can be repeated to wash the adipose tissue sample. The second operation (operation 120) of this method shown in FIG. 1A is to dissociate adipose tissue. Adipose tissue can be dissociated using ultrasound. Adipose tissue can be dissociated using a dissociation solution. The dissociation solution may contain enzymes that break down excess cellular matrix of adipose tissue. The dissociation solution may comprise collagenase, proteinase, proteinase, neutral protease, elastase, hyaluronidase, lipase, trypsin, liberase, DNase, deoxyribonuclease, pepsin, or combinations thereof. The dissociation solution can range from 0.1 mg / ml to 10 mg / ml (eg, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.5 mg / ml, about 0.75 mg / ml). ml, about 1 mg / ml, about 1.2 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 7 mg / ml, or about 10 mg / ml ml) at a concentration of collagenase. The dissociation solution can contain trypsin. The dissociation solution can contain collagenase and deoxyribonuclease. The dissociation solution can include collagenase, hyaluronidase, and deoxyribonuclease. The dissociation solution may comprise collagenase in the range of 0.2 mg / ml to 5 mg / ml, hyaluronidase in the range of 0.1 mg / ml to 4 mg / ml, and deoxyribonuclease in the range of 1 unit to 400 units. One unit can be defined as the enzyme activity that causes a change in A260 of 0.001 per ml per ml at 25 degrees Celsius and pH 5.0 using DNA as a substrate at a concentration of 4.2 mM Mg ++. The dissociation solution may further contain calcium ions and / or magnesium ions. The dissociation solution has a concentration in the range of 0.1 mM to 10 mM (eg, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.5 mM, about 0.7 mM, about 1 mM, about 1.5 mM, about 2 mM, About 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, or about 10 mM) magnesium ion or calcium ion.

  After adding the dissociation solution, the adipose tissue can be cultured at a specific temperature (eg, about 37 degrees Celsius) and a specific time period (eg, about 5 minutes to about 30 hours). During culture, adipose tissue and dissociation solution can be mixed intermittently and / or continuously to support an efficient reaction. The adipose tissue dissociation or culturing operation is at 37 degrees Celsius and ranges from about 10 minutes to about 120 minutes (about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 60 minutes, 75 minutes, about 90 minutes, or about 120 minutes), the adipose tissue sample in the dissociation solution can be gently agitated continuously or intermittently. The frequency of stirring exceeds every 3 minutes (for example, every 1 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 operation, a metal ion chelator such as ethylenediaminetetraacetic acid (EDTA) is added to the sequestering metal ion to stop the activity of the enzyme in the dissociation solution and the temperature ranges from about 4 degrees Celsius to 30 degrees Celsius. (Eg, 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 can be maintained after incubation, for example at room temperature of approximately 25 degrees Celsius or in the range of 18 degrees Celsius to 28 degrees Celsius. In other embodiments, plasma, platelet rich plasma, or serum can be added after the dissociation operation to inhibit the enzyme in the dissociation solution.

  A third operation (operation 130) of the method of FIG. 1A is to remove free oil, matrix fibers, tissue debris, and unwanted cells such as fat cells. Adipocytes, matrix fibers, and tissue residues are substantially in the range of about 10 μm to 70 μm (eg, 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 60 μm, or It can be removed using a mesh strainer containing pores having a pore size of about 70 μm). In other embodiments, the dissociated adipocyte sample has pores ranging from about 20 μm to about 50 μm (eg, about 20 μm, about 22 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, and about 50 μm). It can be passed through a mesh having dimensions. Filtrate passing through the mesh strainer can be collected. Alternatively, adipocytes, matrix fibers, and tissue debris can be substantially removed using centrifugation.

  The fourth, fifth, and sixth operations (operations 140, 150, and 160) of the method of FIG. 1A reduce red blood cells (RBC), concentrate the cells of interest, and wash the cells, respectively. Including that. The order of these three operations can be interchangeable. For example, in one embodiment, the cells may be first concentrated and washed, followed by RBC reduction. In other embodiments, the cells may be first washed and then concentrated. In still other embodiments, the cells may be washed and concentrated at the same time. The concentration operation can be advantageously performed in applications where a smaller volume is desired. For example, culturing cells isolated from adipose tissue is often performed in research. Cells can be concentrated to increase seeding cell density in cell culture flasks and to increase culture efficiency. Isolated cells can be used for transplantation or infusion into humans or animals. In that case, the advantage that concentrating the cells where a high cell concentration is often desired substantially results in the removal of reagents used in previous operations (eg enzymes in the dissociation solution) for better results. Can also be brought about. Such reagents can inhibit cell growth or have deleterious consequences when implanted in animal or human patients. In one embodiment, a microfluidic device can be used to concentrate the cells of interest and / or to remove red blood cells. The microfluidic device may be configured to accomplish the fourth operation, the fifth operation, and / or the sixth operation simultaneously. Furthermore, the microfluidic device can be configured to remove lymphocytes to reduce the likelihood of an immune response. In other embodiments, the concentration operation can be accomplished using centrifugation. In still other embodiments, the concentration operation can be accomplished using a membrane filter. In yet other embodiments, the concentration operation can be accomplished using hollow fibers (eg, hollow fiber membrane filters). In still other embodiments, the red blood cell reduction operation can be accomplished using a red blood cell lysate (eg, ammonium chloride, etc.). In such a solution, red blood cells are selectively lysed.

  The sixth operation (operation 160) of the embodiment shown in FIG. 1A is to wash the target cells and / or to transfer the target cells to the desired buffer. Centrifugation, buffer exchange, and / or dialysis methods can be used. Alternatively, a microfluidic device can be configured to perform this operation. This procedure can further reduce residual reagents used in previous procedures and may be desirable when the subject cells are used for clinical transplantation. However, in some embodiments, operations 140-160 may be omitted.

  In one embodiment of the present disclosure, the method includes preconditioning adipose tissue, dissociating the adipose tissue, and purifying the released cells. A flowchart of this embodiment of this method, indicated generally by the reference numeral 200, is shown in FIG. 1B. In one embodiment, the adipose tissue preconditioning operation (operation 210) includes draining the waste fluid, removing the waste fluid, removing excess fluid, washing the adipose tissue sample, Washing and / or fragmenting the adipose tissue sample. Fragmentation can be advantageous when the adipose tissue sample contains large chunks that are difficult to digest or dissociate with enzymes. Adipose tissue preconditioning operations can be accomplished using aspects and embodiments for separating non-adipocyte populations from adipose tissue disclosed in the present disclosure. For example, the adipose tissue preconditioning operation can include holding an adipose tissue sample in the first container while passing an excess fluid (such as blood, a tumescent solution, or other body fluid) through the second container. Retention of the adipose tissue sample can be achieved by using a mesh in the first container. In one embodiment, the mesh has a pore size ranging from about 70 μm to about 300 μm (eg, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm, about 200 μm, or about 300 μm). Alternatively, retention of the adipose tissue sample can be accomplished using a detector or sensor that detects the position of the adipose tissue sample relative to the first container without using a mesh in the first container. The adipose tissue preconditioning operation can further include adding and removing a wash solution once or repeatedly to wash the adipose tissue sample. Tissue preconditioning can also be achieved using centrifugation. In this case, the adipose tissue sample is separated from excess fluid and / or waste fluid under centrifugal force. Alternatively, the adipose tissue preconditioning operation may be omitted if the adipose tissue is in an acceptable situation for dissociation and purification.

  In one embodiment, the adipose tissue dissociation operation (operation 220) comprises at least one enzyme (eg, collagenase, protease, proteinase, neutral protease, elastase, hyaluronidase, lipase, trypsin, papain, liberase, deenase, deoxyribonuclease , Pepsin, or combinations thereof) at a temperature suitable for enzymatic digestion (eg, in the range of about 32 degrees Celsius to about 38 degrees Celsius) and a period of time in the range of about 3 minutes to 20 hours. Including culturing. In other embodiments, the adipose tissue dissociation operation comprises passing ultrasound through the adipose tissue sample. Cells are released from the adipose tissue sample during the adipose tissue dissociation operation.

  Purification of released cells (operation 230) can be performed using filters, meshes, hollow fibers, antibodies, microfluidic devices, or centrifuges, cell concentration, cell washing, cell sorting, cell separation, residue removal, non-target It may include cell removal, red blood cell reduction, or a combination of these manipulations. For many applications, such as nursing and field applications, the entire method of the present disclosure is in a short time period (eg, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, or 120 minutes) It would be desirable to be implemented.

  Numerous procedures and embodiments for separating non-adipocyte populations disclosed herein from adipose tissue can be used in research studies and pharmacological test systems. Separation of non-adipocytes can be performed for physiological, metabolic and functional studies, or drug testing in a pharmacological test system. Numerous operations and embodiments disclosed herein can also be used in regenerative medicine for transplantation. When cells obtained using the methods and / or devices disclosed herein are cultured in vitro, in vitro cultured tissue for transplantation can be formed. Adipose-derived stem cells can be isolated to produce functional cells and tissues.

  One embodiment of the present disclosure is a sample processing apparatus generally indicated by reference numeral 300 in FIG. 2A. The sample processing apparatus 300 includes a sample conditioning chamber 310 and a waste chamber 320. The sample conditioning chamber 310 has a first inlet 305 for receiving a sample 340 (eg, aspirated adipose tissue or adipose tissue sample from liposuction) and a wash solution (eg, buffer, saline solution, etc.). A second inlet 315 for receiving from the source 350, a first outlet 325 for collecting the conditioned sample 360, and a second outlet 335 connected to the waste container 320 are included. Second outlet 335 may include means 345 (eg, a valve, pinch valve, stopcock, etc.) for opening or closing the fluid connection between conditioning chamber 310 and waste container 320. The sample conditioning chamber 310 allows the sample to drain excess fluid into a waste container and be cleaned using a cleaning solution. The conditioning chamber has a sensor, preferably proximate to the second outlet 335, for detecting whether the sample is approaching the outlet when excess fluid or wash solution is discharged to the waste container. And further include. The conditioning chamber may further include a strainer configured between the first inlet 305 and the second outlet 335 configured to retain adipose tissue during sample cleaning, during sample cleaning, and during excess fluid removal. . In some embodiments, the strainer ranges from about 30 μm to about 1 mm (eg, about 30 μm, about 50 μm, about 70 μm, about 85 μm, about 100 μm, about 120 μm, about 140 μm, about 200 μm, about 300 μm, about 500 μm, about 700 μm). Or a filter including pores having a pore size of about 1 mm). In other embodiments, the strainer may include pores having pore sizes in the range of about 70 μm to about 500 μm (eg, about 70 μm, about 100 μm, about 140 μm, about 200 μm, about 300 μm, or about 500 μm). The strainer may include a mesh filter having a pore size ranging from about 70 μm to about 200 μm (eg, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm, or about 200 μm). In other embodiments, the strainer has a smaller pore size than the fat tissue piece so that the fat tissue piece is retained by the strainer.

  The cleaning solution may enter the conditioning chamber via a flow controller 330 (eg, a peristaltic pump) that controls the volume of cleaning solution applied to the conditioning chamber. The flow control device may include at least one valve and a variable volume container. For example, the flow control device can include a stopcock 370 and a syringe 380, as shown schematically in FIG. 2B. In order to dispense the measured volume, the stopcock is first switched to a position where the inlet is fluidly connected to the syringe. Pulling the syringe plunger draws fluid from the inlet to the syringe. The stopcock is then switched to a position where the outlet is fluidly connected to the syringe. Next, when the plunger of the syringe is pushed, the fluid is discharged from the syringe through the outlet. When the stopcock is finally switched again, the syringe is fluidly connected to the inlet and the pump cycle is complete. The pump cycle can be repeated until the desired volume of wash solution is added to the conditioning chamber. The flow control device may also include two check valves (ie, valves that allow fluid flow in one direction) CV1 and CV2, and a syringe schematically shown in FIG. 2C. To dispense the measured volume, the plunger of the syringe is first pulled and subsequently pushed to complete the pump cycle. The first check valve (CV1) is configured to allow fluid to flow from the inlet to the syringe, and the second check valve (CV2) is configured to allow fluid to flow from the syringe to the outlet. The The pump cycle includes pushing and pulling the plunger to push the fluid into and out of the syringe, respectively, and can be repeated until the desired volume of wash solution is added to the conditioning chamber. . Alternatively, the syringe in the flow control device shown in FIG. 2B or 2C can be replaced with a bag that can be inflated and deflated, or a container that can change volume in a controlled manner.

  Another embodiment of the present disclosure is a sample processing apparatus, generally indicated by reference numeral 400 in FIG. 2D. The sample processing apparatus 400 includes a sample dissociation chamber 410 and a cell purification apparatus 420. The dissociation chamber has a first inlet 405 for receiving the sample, a second inlet 415 for receiving the dissociation solution from the dissociation solution source 430, and at least one outlet 425 fluidly connected to the cell purification device. And including. The dissociation chamber is configured to dissociate the sample into smaller components (eg, single cells or small cell clumps). The dissociation solution may contain an enzyme that degrades the sample. For example, the dissociation solution can include collagenase, proteinase, proteinase, neutral protease, elastase, hyaluronidase, lipase, trypsin, liberase, DNase, deoxyribonuclease, pepsin, or combinations thereof. The temperature is controlled, for example, to about 37 degrees Celsius, and the sample and fluid in the dissociation chamber can be mixed to support an effective enzymatic reaction and uniform dissociation. One embodiment of the dissociation chamber is a flexible bag. Mixing can be aided by massaging, squeezing, rolling, rocking, shaking, repeating partial squeezing and relaxation, or otherwise stimulating the bag. The dissociation solution can also include a detergent (eg, Tween 20 or sodium dodecyl sulfate). When ultrasound is used to dissociate the sample, the dissociation solution can include a medium that effectively conducts ultrasound. The dissociation chamber may include a strainer (such as a mesh or filter). The strainer may function to hold the sample in one position for effective dissociation and / or remove large residues from the dissociated sample. In some embodiments, the strainer ranges from about 30 μm to about 1 mm (eg, about 30 μm, about 50 μm, about 70 μm, about 85 μm, about 100 μm, about 120 μm, about 140 μm, about 200 μm, about 300 μm, about 500 μm, about 700 μm). Or a filter including pores having a pore size of about 1 mm). In other embodiments, the strainer may include pores having pore sizes in the range of about 70 μm to about 500 μm (eg, about 70 μm, about 100 μm, about 140 μm, about 200 μm, about 300 μm, or about 500 μm). The strainer may include a mesh filter having a pore size ranging from about 70 μm to about 200 μm (eg, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm, or about 200 μm). In other embodiments, the strainer has a smaller pore size than the fat tissue piece so that the adipose tissue is retained by the strainer.

  The cell purification device is connected to the dissociation chamber. In some embodiments, the sample processing device further includes a valve 435 between the dissociation chamber and the cell purification device. Closing this valve allows the sample to be cultured with the dissociation solution, and opening this valve may allow the released cells to enter the cell purification apparatus.

  The cell purification device is configured to receive the released cells from the dissociation chamber and to purify the released cells. In some embodiments, the cell purification device includes a chamber fluidly connected to the dissociation chamber via an inlet 445, an outlet 455 for collecting purified cells 440, and in the dissociated sample. And a strainer configured to remove a large amount of residue. The strainer includes a filter having a pore size ranging from about 10 μm to 100 μm (eg, 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). obtain. The strainer may also include a mesh having a pore size ranging from about 20 μm to 60 μm (eg, 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 apparatus, schematically illustrated in FIG. The sample processing apparatus 500 includes a first chamber 510, a waste container 520, and a cell purification apparatus 530. The cell purification device can include the mesh disclosed above. The first chamber can function as a preconditioning chamber in which the sample can be washed prior to dissociation, and a dissociation chamber in which the sample can be dissociated into smaller components (eg, single cells or small cell clumps). The first chamber includes a first inlet 505 for receiving a sample and a second inlet 515 for receiving a cleaning solution from a cleaning solution source 350. In some embodiments, the wash solution and the dissociation solution may enter the first chamber via the same inlet. In other embodiments, the first chamber includes a third inlet 525 for receiving the dissociation solution from the dissociation solution source 430. The first chamber may further include a strainer (eg, a mesh or filter) as described in the previous paragraph with respect to the dissociation chamber and the conditioning chamber. The first chamber is fluidly connected to the waste chamber and the cell purification device. Valves V1 and V2 (which may include, for example, pinch valves or clamp valves) may be used to control fluid flow. For example, during preconditioning of the sample, the valve V1 can be opened so that excess fluid and wash solution can flow from the first chamber into the waste container. During sample dissociation, both V1 and V2 can be closed so that the sample can be incubated with the dissociation solution. After dissociation, V2 can be opened so that the dissociated sample can be transported to the cell purification device.

  In some embodiments of the present disclosure, the adipose tissue sample is pre-heated to a specific temperature (eg, 37 degrees Celsius) before being loaded into the first chamber 510. Pre-heating as a treatment of adipose tissue sample can reduce the time required to process the tissue sample. In other embodiments of the present disclosure, the adipose tissue sample is photoactivated with light having an average wavelength in the range of 300 nm to 700 nm before being loaded into the first chamber 510. Photoactivation can increase the effectiveness of the cells in the tissue sample. In yet another embodiment of the present disclosure, the adipose tissue sample is processed using acoustic waves (ie, sound waves that relax the tissue and facilitate dissociation of the tissue) before being loaded into the first chamber 510. The

  The sample processing device may further include a flow control device 330 as described above to control the flow of cleaning solution into the first chamber. The flow controller 330B (which can be similar to the flow controller 330) can also be used to control the flow of dissociation solution injected into the first chamber. In some embodiments, the dissociation solution is loaded into a syringe before being injected into the first chamber.

  It will be appreciated that the sample processing apparatus disclosed herein has different variations and combinations. For example, as shown in FIG. 2F, an embodiment of the sample processing apparatus, generally designated by reference numeral 500A, includes two to control fluid flow between the first chamber, the waste container, and the cell purification apparatus. Valves (V1 and V2) may be used. The flow control device may include two check valves (CV1 and CV2) and a volumetric device 540 (eg, a syringe). The dissociation solution can be connected to the first chamber via a check valve CV3.

  Another embodiment of the sample processing apparatus of the present disclosure is the sample processing apparatus schematically illustrated in FIG. In this sample processing apparatus, at least three stopcocks (SC1, SC2, and SC3) are used to control the fluid flow. For example, excess fluid and wash solution can be transferred from the first chamber to a waste container while preconditioning the sample. When the stopcock (SC3) closes off the flow exiting the first chamber, the sample can be incubated with the dissociation solution during sample dissociation. After dissociation, the stopcock (SC3) can connect the first chamber to the cell purification device so that the dissociated sample can be transported to the cell purification device.

  FIG. 3A shows an embodiment of the apparatus schematically shown in FIG. 2E. The device includes two plastic sheets. These plastic sheets are joined together, thereby forming a plurality of chambers. This apparatus can support the method for adipose tissue processing disclosed herein in a smooth, easy to use, safe and cost effective manner. Chamber 1 is a measurement chamber that can be expanded to a specific volume. The chamber includes an inlet (port 1) and an outlet (port 2). The chamber 1 takes in fluid and discharges a predetermined volume of fluid so that the volume of fluid discharged into the chamber 2 through the inlet (port 3) for adipose tissue processing is controlled. Can be configured. Port 2 and port 3 can be fluidly connected by a single tube. When the tube is tightened using a pinch valve, clamp valve, stopcock, check valve, or other valve mechanism (s), port 2 and port 3 may be fluidly disconnected. In order to operate chamber 1, the connection between port 2 and port 3 is initially disconnected. As fluid is introduced into chamber 1 via port 1, chamber 1 is fully or partially inflated. Port 1 is then closed using, for example, a pinch clamp, pinch valve, stopcock, check valve, or other valve mechanism (s). Next, the valve between port 2 and port 3 is opened. Thereby, the fluid in the chamber 1 can flow into the chamber 2 when the chamber 1 is contracted. Shrinkage of chamber 1 can be achieved by externally squeezing and / or compressing chamber 1 using gravity, siphoning, or other methods well known in the art. Chamber 1 can be placed higher than chamber 2 in order to assist in discharging fluid into chamber 2 using gravity. A predetermined amount of fluid is transferred to the chamber 2 by this process. Such an amount is determined by the difference between the expansion volume and the contraction volume of the chamber 1. If a smaller amount of fluid is desired, the chamber 1 can be partially compressed, squeezed, and / or tightened so that the volume of fluid entering and / or exiting the chamber 1 is controlled. Alternatively, the chamber 1 can only be partially deflated so that a portion of the fluid in the chamber 1 is discharged. If a larger volume is desired, the expansion / contraction process can be repeated several times until the desired volume of fluid is delivered to the chamber 2.

  Chamber 1, port 1 and port 2 can be an embodiment of the flow control apparatus schematically shown in FIG. 2B or 2C.

  Port 1 and port 2 may each include a check valve, also known as a check valve, that only allows fluid to enter and exit chamber 1. The operation of measuring and discharging a predetermined volume of fluid is very simple. That is, when the chamber 1 is expanded, the fluid can enter through the port 1, and when the chamber 1 is compressed, the fluid can be pushed out through the port 2.

  Alternatively, chamber 1 can be a storage chamber. Providing a storage chamber provides the prepackaged solution needed for sample processing. For example, lactate Ringer's solution, buffered saline solution, physiological saline solution, dissociation solution, washing solution, washing solution, a solution containing ethylenediaminetetraacetic acid (EDTA), and / or an enzyme solution are contained in the chamber 1 as part of the apparatus. Can be packaged.

  In other embodiments, the measurement chamber may include a syringe that includes a plunger. Such syringes can draw and / or dispense a predetermined volume of fluid by withdrawing and pushing the plunger. In still other embodiments, the measurement chamber can include a flexible tube mounted on a peristaltic pump. Here the fluid flow can be controlled using a peristaltic pump.

  Chamber 2 can be an embodiment of the first chamber 510 shown schematically in FIG. 2E. Chamber 2 can be an adipose tissue cleaning chamber that includes one or more inlets and outlets. Chamber 2 may also act as a dissociation chamber and may further include at least one mesh (eg, a screen, a semi-permeable membrane, and / or a filter mechanism including a porous or microporous membrane). Adipose tissue samples (such as aspirated adipose tissue or aspirated adipose tissue pieces) can be introduced or loaded into the chamber 2 via the inlet (port 4). Excess fluid from the sample can be drained through the mesh via connector 1 to the waste collection chamber (chamber 3). A cleaning solution can be added to the chamber 2 to clean and / or wash the sample. The cleaning solution can be measured and discharged through chamber 1 into chamber 2. Solutions for processing the sample can be added to change the sample. Mixing means can be added to the chamber 2 so that cleaning and cleaning are more effective. For example, chamber 2 may be massaged, gently squeezed, rocked, shaken, repeated partially squeezed and relaxed, or otherwise stimulated to assist in mixing. obtain. The waste fluid can then be discharged to a waste collection chamber (chamber 3). The outlet of the chamber 2 (connectors 1 and 2) can be closed using valve means during mixing to allow complete mixing between the cleaning solution and the sample before the waste fluid is drained. . Clamps (eg, clamps 1, 2, and / or claim 3 shown in FIG. 3F) may be added to close the fluid connection between the chambers by tightening these chambers. The washing process can be repeated several times (eg, 2, 3, 4 or 5 times). During the washing operation, the adipose tissue sample can be retained in the chamber 2. For adipose tissue samples containing small agglomerates, chamber 2 may contain a mesh or membrane filter so that the sample is effectively retained. Multiple mesh layers and / or filter layers (Mesh 1 and Mesh 2) may be incorporated to provide the desired adipose tissue retention.

  After washing, a dissociation solution can be added to chamber 2 so that the adipose tissue sample is dissociated and cells are released. The temperature of the chamber 2 can be set to a specific optimum temperature using heating, cooling, and / or temperature control systems to assist in sample dissociation. For example, the device may be placed in an incubator, a water bath, and / or placed in contact with a constant temperature plate so that the temperature is maintained at about 37 degrees Celsius for optimal enzyme digestion. Good. The dissociation solution may contain one or more enzymes to degrade the binding matrix, extracellular matrix, etc. For example, collagenase can be used at 37 degrees Celsius to degrade collagen fibers. Other reagents (including proteinase, protease, trypsin, proteinase K, lyase, enzyme, lysate, hyaluronidase, lipase, trypsin, liberase, DNase, deoxyribonuclease, pepsin, or combinations thereof) for adipose tissue digestion Can be used. The outlet (connector 1 and / or connector 2) in the dissociation chamber (chamber 2) can be closed during digestion. Chamber 2 is massaged, gently squeezed, rocked, shaken, repeated partial squeezing and relaxation to facilitate mixing and promote effective adipose tissue dissociation, or Different stimulation can be performed. When digestion is complete, connector 2 is opened and the released cells can exit the dissociation chamber. At least one mesh in the chamber 2 may act to remove or retain large residues. One or more chase wash operations involving the addition of a wash solution can be applied to wash cells that may be trapped in the chamber 2 after digestion.

  The sample processing apparatus shown in FIG. 3A may further include a residue removal chamber (chamber 4) that may include a mesh, membrane filter, and / or other residue removal mechanism (eg, mesh 3). The dissociated sample can be transported to a residue removal chamber. In the residue removal chamber, large clumps, unwanted cells (such as adipocytes), and / or residues can be removed from the sample. Chamber 4 can also act as a sample storage chamber. In the sample storage chamber, the released cells are stored until the next use. Released cells can be collected via port 5. Chamber 4 may be an embodiment of a cell purification device 530 shown schematically in FIG. 2E.

  3B and 3C show an embodiment in which the mesh is incorporated into the chamber 4. The chamber 4 can be formed by folding the mesh, sandwiching it between two flexible sheets, and welding, bonding, heat fusing, or otherwise fusing. Similarly, a mesh or membrane filter can be incorporated into the chamber 2. FIGS. 3D and 3E each include an exploded view and a cross-sectional view of a portion of chamber 2, showing that one or more meshes can be used for chamber 2. The mesh contained in chamber 2 and / or chamber 4 may include pores that become progressively smaller along the fluid flow path through chamber 2 and / or chamber 4. For example, the nominal pore size of mesh 1 (FIG. 3D) can range from about 100 μm to about 900 μm, the nominal pore size of mesh 2 can range from about 50 to about 200 μm, and the mesh 3 (FIG. 3C) The nominal pore size can range from about 10 μm to about 60 μm.

  It will be understood that the present disclosure is not limited to the particular configuration of the embodiment shown in FIGS. 3A-3F. Embodiments of the present disclosure may include a greater number of chambers or fewer chambers than the number of chambers illustrated in FIGS. Chambers with various functions can be used, and such chambers can be configured to accomplish specific tasks including a specific sequence of operations. For example, these chambers can be used for sample collection, washing, washing, stratification, mixing, heating, cooling, filtration, digestion, storage, valve operation, volumetric measurement, pumping, fluid transport and manipulation, cell labeling, sample processing , Dissociation, waste fluid collection, aggregate removal, residue removal, lysis, concentration, polymerase chain reaction (PCR), culture, hybridization, cell culture, cell expansion, etc.

  Another embodiment of the sample processing apparatus of the present disclosure, generally indicated by the reference numeral 600 in FIG. 4, is formed using two plastic sheets. Chamber 1 is a sample washing / dissociation chamber, and includes three inflow ports (port 1, port 2, and port 3), a mesh (mesh 1), and two outflow connectors (connector 1 and connector 2). Including. Chamber 2 is an agglomerate reduction chamber and includes an inlet connector (connector 2), an outlet / inlet connector (connector 3), and a mesh (mesh 2). If desired, the chamber 3 containing the mesh (mesh 3) can be a reservoir for separated cells and even a residue removal, or a cell purification chamber. Chamber 4 is a waste solution collection chamber and includes an inlet tube (connector 1) in fluid communication with the outlet connector of the sample cleaning / dissociation chamber (chamber 1).

  5A and 5B illustrate a chamber 610 of another embodiment of the present disclosure that includes two meshes. The two meshes are configured so that they do not substantially overlap. Each mesh is folded and sandwiched between two flexible outer sheets. This embodiment may have the advantage of being easy to fabricate since only a two layer mesh is required to be fused between the sheets.

  6A and 6B show a chamber 620 of yet another embodiment of the present disclosure that includes at least one unfolded mesh sandwiched between two flexible outer sheets. The mesh is disposed between the inflow port and the outflow port and is configured such that fluid entering from the inflow port must pass through the mesh to reach the outflow port. The inflow port and the outflow port are on opposite sides of the mesh. This configuration can have the advantage of being easy to fabricate since only a single layer of mesh is required to be sealed between the two sheets.

  FIG. 7 shows a chamber 630 of yet another embodiment of the present disclosure that includes a pleated mesh sandwiched between two flexible outer sheets. This configuration can have the advantage of a large mesh area.

  8A and 8B show a chamber 640 of yet another embodiment of the present disclosure. Here, before being incorporated into the chamber, the mesh is folded and sealed along the sealing edge, thereby forming a pouch. One plastic mesh (eg polyamide mesh) is folded along the fold line and sealed along the sealing line using, for example, heat sealing or high frequency welding, thereby forming a mesh pouch. The mesh pouch is then placed between two flexible plastic sheets (eg, polyvinyl chloride (PVC) sheets, etc.) and sealed along the sealing periphery using, for example, heat sealing or radio frequency welding. Can form a chamber.

  9A and 9B illustrate a chamber 650 of yet another embodiment of the present disclosure. Here, the mesh is folded and sandwiched between two flexible plastic sheets and sealed to the chamber. Here, in the chamber and the mesh, the sample entering the chamber via the inflow port flows out through the outflow port 1 without passing through the mesh, while the portion of the sample flowing out through the outflow port 2 passes through the mesh. Configured to be. The mesh fold line is substantially vertical. In other embodiments, the fold line may form a constant angle with respect to the vertical.

  One or more mesh configurations shown in FIGS. 5A-9B may be used with any chamber of any sample processing apparatus disclosed herein (eg, one of chamber 2 or chamber 4 of sample processing apparatus 500). One or more and / or one or more of chamber 1, chamber 2, and / or chamber 3 of sample processing apparatus 600).

  10A and 10B show the connector between the two chambers. Such a connector may include at least one tube portion that may be included in any embodiment of the sample processing apparatus disclosed herein. The plastic sheet is cut and the tube is bridged across the cut from one chamber to the other. This configuration may allow the valve means to access the tube. For example, pinch clamps or slide clamps can be used to interrupt the fluid connection through the tube. The tube portion further includes a section having flexibility and flexibility (tube 2 in FIGS. 10A and 10B) to assist in reliable tightening. This can be an advantage when a pinch valve is used. The pinch valve can be actuated manually, pneumatically, or using a solenoid. The flexible tube can support peristaltic pump operation when needed.

  Embodiments of the sample processing apparatus disclosed herein include other portions shown in FIG. 11 (eg, a spike that can be inserted into a cleaning solution bag, one or more pinch clamps, a Y-shaped insertion site, a spike port, and And / or a luer connector for connecting to a syringe) and / or connected to other modules as an integrated part of a larger system. Bulkheads, injection ports, spike ports, valves, check valves, tubes, adapters, lures, female luer locks, male luer locks, syringes, stopcocks, and / or other connection mechanisms are part of the embodiments of the present disclosure. It can be used to interconnect other parts.

  Another embodiment of the present disclosure is indicated generally by the reference numeral 700 in FIG. 12, including a sample dissociation chamber (chamber 1), a waste container (chamber 2), and a cell purification chamber (chamber 3). It is a sample preparation device. Chamber 1 may optionally include a first filter mesh to assist in sample cleaning, washing, and preconditioning. The mesh includes pores having a pore size ranging from about 20 μm to about 600 μm. In processing aspiration adipose tissue samples, the mesh may range from about 40 μm to about 200 μm (eg, 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 140 μm, about 170 μm). Or about 200 μm) would be preferred. The stopcock controls the fluid connection at port 5. This stopcock is closed while sample dissociation and incubation is performed, and is connected to chamber 2 via port 7 while sample wash is performed, and port 6 is connected to collect released cells. To the chamber 3. Chamber 3 may include a second filter mesh having a pore size ranging from about 15 μm to about 150 μm. The second mesh removes residues from the dissociated sample and purifies the released cells. Purified cells can be collected at port 8. The apparatus includes an opening for receiving a sample (eg, port 4) (this opening may include a spike port) and an inlet (eg, port 1) for receiving a cleaning solution (this inlet includes a spike). And at least one inlet (eg, port 2) for receiving the dissociation solution.

  Yet another embodiment of the present disclosure was adhered in a predetermined pattern to form a sample dissociation chamber (chamber 1), a waste container (chamber 2), and a cell purification chamber (chamber 3). FIG. 14 is a sample preparation device, generally designated by the reference numeral 800 in FIG. 13, comprising two flexible sheet materials (eg, plastic). Chamber 1 may include a first mesh (mesh 1) to assist in sample cleaning, washing, and preconditioning. Chamber 3 may include a second mesh (mesh 2) having a pore size smaller than the pore size of the first mesh. Stopcock 1 controls the fluid connection between chamber 1, chamber 2, and chamber 3. Chamber 1 includes an inflow port (port 1) that includes a connector that assists in receiving a sample from a syringe (eg, a syringe having a catheter tip). The chamber 1 further includes another port (port 2) connected to a stopcock manifold including the stopcock 2 and the stopcock 3. A washing solution (eg Lactated Ringer's Injection) can be connected to the sample preparation device via a spike. The syringe 1 and stopcock 2 can work together to act as a flow control device that allows a defined amount of cleaning solution to be added to the chamber 1. The dissociation solution can be loaded into the syringe 2 and added to the sample preparation device via the stopcock 3. The dissociation solution can be loaded into the syringe 2 in concentrated form and reconstituted to a normal working concentration using a wash solution. Chamber 3 includes an outflow port where the released cells are collected. In some embodiments, it is desirable to increase the pressure at the outflow port. Chamber 3 can be sealed in an airtight manner in a pressurized chamber (chamber 4). Chamber 4 includes a pressure port. At this pressure port, pressurized fluid (eg, compressed air) can be applied to pressurize the fluid in chamber 3 indirectly through the flexible sheet of chamber 3. An embodiment of chamber 3 is shown in FIG. 13B, and an embodiment of a pressurized chamber that includes chamber 4 is shown in FIG. 13C. Chamber 3 is formed by joining two flexible sheets together at a predetermined location (FIG. 13B). A cut (cut 1) is made in the sheet, which allows another sheet to seal chamber 3 and form chamber 4.

  Another embodiment of the present disclosure includes a downstream processing unit (DPU 1000 shown in FIG. 14). The downstream processing unit may further process, purify, culture, expand, and / or analyze the adipose tissue and / or the separated cells. The downstream processing unit has the following functions (for example, sample washing, sample concentration, sample fractionation, sample concentration, sample separation, buffer exchange, sample labeling, sample alteration, filtration, magnetic labeling, magnetic fractionation, polymerase chain reaction) (PCR), antibody interaction, affinity capture using antibodies, cell imaging, enzyme-linked immunosorbent assay (Elisa), protein preparation, protein purification, protein concentration, mass spectrometry, high performance liquid chromatography, flow Cytometry, cell classification, functional assay, cell culture, cell expansion, cell differentiation, immunophenotyping, lateral flow assay, fluorescence in situ hybridization, deoxyribonucleic acid (DNA) hybridization, ribonucleic acid (RNA) hybridization, deoxyribonucleic acid (DNA) reaction, Bo acid (RNA) reaction, it may be configured to support one or more of the other, etc.).

  The downstream processing unit may include a microfluidic unit that includes at least one microfluidic device. The microfluidic device has at least one channel dimension smaller than 1 mm (e.g., 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 include at least one substantially constant depth channel. For example, the microfluidic device is 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 a channel having a depth of about 15 μm. The channel depth of the microfluidic device can be in the range of 20% of the nominal channel depth. The microfluidic device can further include a channel on substantially one surface that can be substantially flat or curved. The microfluidic device may also include a channel formed on one or more substantially flat surfaces.

  Microfluidic devices include optical lithography, etching, reactive ion etching, deep reactive ion etching, wet etching, imprinting, injection molding, embossing, soft embossing, stereolithography, molding, soft lithography, anodic bonding, super It may be formed using micromachining, nanomachining, and / or micromachining techniques including but not limited to sonic bonding, self-assembly, and / or other fabrication techniques well known in the art.

  Embodiments of the microfluidic unit of the present disclosure are described in International Application PCT / US10 / 061866, International Publication No. 2011/079217 (A1), US Pat. No. 7,150,812 (B2), US Pat. No. 7,735. , 652, U.S. Patent No. 8,021,614, U.S. Patent No. 8,186,913 (B2), U.S. Patent Application Publication No. 2012/0063664 (A1), and U.S. Patent Application Publication No. 2011/0294187 ( A1) apparatus (these patents are hereby incorporated by reference for all purposes) and Dean flow, inertial force, centrifugal force, deterministic lateral displacement, strut array , Columnar array, pinch flow, magnetic structure, antibody component, cell capture moiety, protein capture moiety, deoxyribonucleic acid (DNA) moiety I, ribonucleic acid (RNA) moiety, filtered, Tangential Flow Filtration, ultrasound focusing, pinch flow, a device to use other, may include.

  It is noted that some embodiments of the present disclosure, particularly those incorporating microfluidic devices disclosed in WO 2011/079217 (A1), provide devices that are resistant to severe clogging and adhesion. Deserve. Traditionally, clogging and adhesion has been a serious problem that prevents the use of microfluidic devices for tangential flow filtration of digested adipose tissue.

  Other embodiments of the present disclosure include a hollow fiber unit to concentrate and / or wash separated cells.

  In another embodiment of a downstream processing unit that includes a microfluidic device, the microfluidic device washes cells and removes unwanted reagents. The downstream processing unit may include a buffer inlet for introducing a buffer for washing the cells. Cell washing can also be achieved using a microfluidic device designed to perform dialysis.

  In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that reduces the enzyme concentration at the outlet of the downstream processing unit to be less than about 1/10. For example, the enzyme concentration is about 1/10, about 1/20, about 1/30, about 1/40, about 1/50, about 1/70, about 1/100, about 1/150, about 1/200, About 1/400, about 1/500, about 1/750, about 1/1000, about 1/2000, about 1 / 5,000, about 1 / 10,000, about 1/20000, Reduced to about 1 / 50,000, about 1 / 100,000, about 1 / 200,000, about 1 / 500,000, or about 1 / 1,000,000. One embodiment of a microfluidic device that can achieve such enzyme removal is disclosed in WO 2011/079217 (A1). According to WO 2011/079217 (A1), the microfluidic device comprises struts and utilizes at least one buffer stream (ie a stream of wash solution) to wash the cells.

  In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that removes greater than 89% of the enzyme 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% of the enzyme introduced during sample dissociation, 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% are removed The

  In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that removes about 100% of the enzyme introduced during sample dissociation.

  In still other embodiments of the present disclosure, the downstream processing unit is less than about 0.1 mg / ml (eg, about 0.09 mg / ml, about 0.05 mg / ml, about 0.03 mg / ml, about 0.02 mg / ml). 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 A) a microfluidic device that provides an output having a collagenase concentration;

  In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that provides an output that is substantially free of enzymes introduced during sample dissociation. In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that provides an output that does not have an enzyme introduced during sample dissociation.

  In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that provides an output that is substantially free of collagenase introduced during sample dissociation. In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that provides an output without collagenase introduced during sample dissociation.

  Examples of microfluidic devices that may be used in one or more of the sample processing devices disclosed herein are generally designated 910, 15A, 15B, 15C, 15D, and 15E, respectively. 920, 930, 940, and 950 are indicated schematically.

  FIG. 15A shows a microfluidic channel 910 having a cell inlet, a buffer inlet, a cell outlet, and a buffer outlet. Since the microfluidic channel has a very small (eg, about 100 μm) width and / or depth, the cell sample and buffer flow in two laminar streams that flow parallel to each other without substantial convective mixing. Form. The flow rate is configured to provide particles (eg, enzymes) that do not require sufficient time to diffuse from the cell stream to the buffer stream. Since cells are much larger than unwanted particles, cell diffusion is very small and remains in the cell stream. The cell stream and buffer stream exit the microfluidic channel via different outlets. Thereby, unnecessary particles are substantially removed.

  FIG. 15B shows another microfluidic channel 920 having a cell inlet, two buffer inlets, a cell outlet, and two buffer outlets. These channels and flow rates are configured to allow unwanted particles to diffuse from the cell stream to the buffer stream. Thereby, unnecessary particles are substantially removed. This configuration can have a high removal efficiency because unwanted particles are removed from the two buffer streams.

  In order to stabilize the cell stream and buffer stream, promote molecular diffusion, and / or support the microfluidic channel, pillars or struts, for example as shown in microfluidic channel 930 shown in FIG. Can be placed.

  Microfluidic devices utilized for dialysis can be configured in series to form a cascade. One case is shown in FIG. 15D. In FIG. 15D, the buffer carrying unwanted particles can be removed from the buffer outlet 1 and fresh buffer can be introduced from the buffer inlet 2. As a result, the remaining unnecessary particles are substantially removed.

  FIG. 15E shows another embodiment of a microfluidic device 950 that includes a channel and a column array within the channel. The flow rate of fluid introduced into the channel and the channel dimensions are designed so that the fluid stream is laminar. Typically, laminar and laminar fluid streams occur when the Reynolds number of the fluid in the microfluidic device is less than about 1. The presence of the column array increases the effective diffusion coefficient of the particles in the buffer stream and improves the efficiency of removing unwanted particles (such as enzymes) from the cell stream.

  In some embodiments, the buffer used to form the buffer stream in the microfluidic device is a wash solution.

  In still other embodiments, the downstream processing unit includes a dialysis membrane.

  In yet another embodiment shown schematically in FIG. 16, the downstream processing unit 1000 is configured to receive at least one microfluidic device that concentrates the separated cells and the separated cells as the output of the microfluidic device. And a microfluidic device unit including at least one collection reservoir (eg, a collection bag). The downstream processing unit may further include a syringe connected to the collection reservoir. After the sample has been processed using the microfluidic device, the output can be withdrawn from the collection reservoir to the syringe. The downstream processing unit may further include a waste storage tank (eg, a waste bag).

  In other embodiments of the present disclosure, the downstream processing unit includes a plurality of microfluidic devices to achieve a desired volume, throughput, and function for processing large output samples.

  Transport of fluid is gravity, external pressure, vacuum, positive pressure, negative pressure, head height, pump (eg, peristaltic pump), mechanism configured to squeeze the bag, roller to squeeze the bag, plate to squeeze the bag And / or other fluid transport mechanisms known in the art. In one embodiment, the fluid can be delivered using a syringe. In other embodiments, the fluid may be conveyed using external air pressure applied to a chamber (eg, chamber 4 wrapping a bag containing cells (chamber 3) as shown in FIG. 13).

  In other embodiments of the present disclosure, the downstream processing unit includes a cell culture chamber for culturing cells. Cell culture chambers can be connected using connectors. Therefore, the cell culture chamber is detachable. The cell culture chamber can be placed in an incubator. Within the incubator, the temperature and conditions for cell growth can be optimized (eg, at about 37 degrees Celsius and about 5% carbon dioxide concentration). The cell culture chamber may further include a material that is permeable to air, such as a filter membrane or a silicone rubber membrane. This allows gas exchange during cell culture.

  One or more chambers of the sample processing apparatus disclosed herein may include a mesh, a multi-layer mesh, and a cascade of meshes (FIG. 5B). In some embodiments, the pore size (eg, median or average pore opening size) of the mesh used for adipose tissue processing ranges from about 1 μm to about 10 mm (eg, about 1 μm, about 3 μm, About 6 μm, about 10 μm, about 15 μm, about 25 μm, about 40 μm, about 70 μm, about 100 μm, about 140 μm, about 300 μm, about 700 μm, about 1 mm, about 2 mm, or about 3 mm). To separate non-adipocytes from aspirated adipose tissue, the mesh pore size can range from about 10 μm to about 2 mm. In some embodiments, meshes with pore sizes of about 40 μm, about 70 μm, about 100 μm, about 140 μm, about 250 μm, and / or about 700 μm (eg, polyamide (nylon) mesh) can be used.

  Other embodiments of the sample processing apparatus of the present disclosure include one or more chambers that include two meshes. Here, the second mesh is in fluid communication with the downstream side of the first mesh, and the pores of the second mesh are substantially smaller than the pores of the first mesh.

  Still other embodiments of the sample processing apparatus of the present disclosure include one or more chambers that include two membrane filters. The second membrane filter can be in fluid communication downstream of the first membrane filter. The pores of the second membrane filter can be substantially smaller than the pores of the first membrane filter.

  Still other embodiments of the sample processing apparatus of the present disclosure include one or more chambers that include membrane filters with etched tracks.

  Embodiments of the sample processing apparatus disclosed herein or their subcomponents include thermoplastics, acrylonitrile butadiene styrene (ABS), acrylic resin (PMMA), celluloid, cellulose acetate, cyclic olefin copolymer (COC) , Cyclic olefin copolymer (COP), ethylene vinyl acetate (EVA), ethylene vinyl alcohol (EVOH), fluororesin (FEP, PFA, CTFE, ECTFE, ETFE, and PTFE), ionomer, liquid crystal polymer (LCP), polyoxymethylene (POM or acetal), polyacrylate (acrylic resin), polyacrylonitrile (PAN or acrylonitrile), polyamide (PA or nylon), polyamideimide (PAI), polyaryl ether Terketone (PAEK or ketone), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polycaprolactone (PCL), polychlorotrifluoroethylene (PCTFE), polyethylene terephthalate (PET), polycyclohexylenedimethylene Terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoate (PHA), polyketone, polyester, polyethylene (PE), polyetheretherketone (PEEK), polyetherketoneketone (PEKK), polyetherimide (PEI), Polyethersulfone (PES), chlorinated polyethylene (CPE), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene Xoxide (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) can be used to construct. For medical applications, flexible plastic sheets (eg, polyvinyl chloride (PVC), polyurethane (PU), ethylene vinyl acetate (EVA), polyamide (PA or nylon), etc.) can be used as the sheet material. In some embodiments, the sample processing apparatus disclosed herein can include two flexible sheets bonded together and having one or more chambers defined therebetween. In other embodiments, the sample processing apparatus disclosed herein may include a flexible sheet. The flexible sheet is bonded to a hard or semi-rigid material (eg, plastic and / or any one or more thick sheets of the materials disclosed above), thereby providing one or more chambers Is defined between the flexible sheet and the hard or semi-rigid material.

  The thickness of the sheet material in some embodiments ranges from about 0.1 mm to about 0.8 mm (eg, 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 sheet material thickness in other embodiments ranges from about 0.2 mm to about 0.4 mm (eg, about 0.2 mm, about 0.25 mm, about 0.3 mm, about 0.35 mm, or about 0.4 mm). It can be.

  Materials for the membrane filter and / or mesh are cellulose acetate (CA), glass microfiber (GMF), polyethersulfone (PES), polypropylene (PP), regenerated cellulose (RC), polyamide (PA or nylon), Including but not limited to polytetrafluoroethylene (PTEE) and / or polyvinylidene fluoride (PVDF).

  The thickness of the mesh member, in some embodiments, ranges from about 10 μm to about 1,000 μm (eg, 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 ). In other embodiments, the thickness of the mesh member ranges from about 50 μm to about 300 μm (eg, 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 include plastic welding, heat sealing, injection molding, embossing, adhesive bonding, ultraviolet light (UV) curable bonding, solvent bonding, hot air welding, freehand welding, speed tip welding, stamping welding, contacts Using standard plastic manufacturing techniques including but not limited to welding, hot plate welding, radio frequency welding, radio frequency welding, injection welding, ultrasonic welding, friction welding, spin welding, laser welding, and / or solvent welding Can be built.

  Embodiments of the present disclosure (eg, sample processing apparatus schematically illustrated in any of FIGS. 2A-2G) can be constructed using high frequency welding of thermoplastic sheets. The welding die can be made of metal (eg, aluminum, brass, or stainless steel). The collapsible mesh piece and the tube piece are placed between two thermoplastic sheets on a welding die. The thermoplastic sheet is then sandwiched using another welding die. A chamber is formed when pressure, temperature, and radio frequency power are applied to the welding die. A range of about 25 degrees Celsius to about 120 degrees Celsius (e.g., about 25 degrees Celsius, about 50 degrees Celsius, about 60 degrees Celsius, about 70 degrees Celsius, 80 degrees Celsius, about 90 degrees Celsius, about 100 degrees Celsius, about 110 degrees Celsius, or about 120 degrees Celsius) and a range of about 10 pounds per square inch to about 600 pounds per square inch (e.g., about 10 pounds / square Square inch, about 20 pounds per square inch, about 30 pounds per square inch, about 40 pounds per square inch, about 50 pounds per square inch, about 60 pounds per square inch, about 80 pounds per square inch, about 100 pounds per square Inch, about 150 pounds per square inch, about 200 pounds per square inch, about 300 pounds per square inch, about 400 pounds per square inch, about 500 pounds per square Inch, or 600 pounds per square inch) and a range of about 300 W to 10 kW (eg, about 300 W, about 500 W, about 600 W, about 700 W, about 800 W, about 900 W, about 1 kW, about 1.2 kW, about 1. Radio frequency power of 5 kW, about 2 kW, about 2.5 kW, about 3 kW, about 4 kW, about 5 kW, about 6 kW, about 7 kW, about 8 kW, about 9 kW, or about 10 kW). Radio frequency power ranges from about 0.5 seconds to about 1 minute (eg, about 0.5 seconds, about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds) , About 8 seconds, about 10 seconds, about 12 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, and about 60 seconds). The radio frequency power can be applied several times with equal or different strengths to form a reliable seal. For medical applications, this device is manufactured in a controlled clean environment and uses standard sterilization techniques including but not limited to gamma irradiation, ethylene oxide (EO) sterilization, and ultraviolet light (UV) irradiation. Can be used to sterilize.

  In some embodiments of the present disclosure, the sample preparation device is sterilized. In some embodiments of the present disclosure, the sample preparation device is a single use. Further, in some embodiments of the present disclosure, the sample preparation device is a substantial protective barrier that provides an isolated environment. In such an isolated environment, in order to minimize or avoid the risk of contamination and infection, the sample is in direct physical contact with the external environment and / or operating personnel, for example through an unfiltered air stream. Or protected from fluid contact.

  It is understood that embodiments of the present disclosure can act as a protective barrier that substantially reduces or eliminates direct physical contact, fluid connection, and / or air flow connection between the adipose tissue sample and the surrounding environment. Is done. Any portion of the device embodiments disclosed herein in direct physical contact with the sample, in fluid connection, and / or in connection with an unfiltered air stream may be sterilized. And / or single use. It will be appreciated that embodiments of the devices disclosed herein may substantially protect the sample from the risk of contamination and protect the operator from the risk of infection.

  It will be appreciated that the present disclosure allows for the design of a very easy to use adipose tissue processing device. It will be appreciated that the present disclosure can dramatically simplify the manufacturing process of such an adipose tissue processing device and reduce the manufacturing cost of such an adipose tissue processing device. According to embodiments of the present disclosure, processing of the adipose tissue sample to be processed is substantially free of contamination and risk of infection by using an apparatus that substantially separates the sample from the surrounding laboratory or hospital environment. It will be further understood that it can be anything.

Example 1 Separation of non-adipocytes from human aspirated adipose tissue Human aspirated adipose tissue was processed using the method comprising the procedure shown in FIG. 1 and the apparatus shown in FIGS. 3A and 11. The device is about 25 cm wide and about 40 cm long. The flexible plastic sheet is made of polyvinyl chloride (PVC), and the mesh is made of polyamide (nylon). Meshes 1, 2, and 3 have nominal pore sizes of about 140 μm, about 70 μm, and about 35 μm, respectively. Approximately 40 ml of human aspirated adipose tissue was collected from approved donors using Tumecent liposuction and processed within 24 hours of collection. Samples were shipped and stored at 4 degrees Celsius prior to processing.

  Initially, clamps 1 and 2 were applied to close connectors 1 and 2 (FIG. 3F). Approximately 100 ml of aspirated adipose tissue was added to the device via a spike port (FIG. 11). When the clamp 2 was opened, excess fluid, including blood and tumecent solution, could be carried out into the chamber 3 under gravity. After the excess fluid was substantially removed, the clamp 2 was closed and the pinch clamp 1 was opened, thereby allowing about 50 ml of lactated Ringer's solution to enter the measurement chamber (chamber 1). The pinch clamp 1 was closed and the pinch clamp 2 was opened. When the chamber 1 was squeezed using two flat plates, the lactated Ringer's solution was transferred to the chamber 2. This fluid measurement and delivery process was repeated until approximately 100 ml of lactated Ringer's solution was added to chamber 2. By gently massaging chamber 2, the Lactated Ringer solution and the aspirated adipose tissue sample were mixed, thereby washing the sample. When the clamp 2 is opened, the waste fluid can be discharged. This washing operation was carried out three times here.

  The tissue dissociation operation was initiated by closing clamps 1 and 2 and adding the dissociation solution from the Y-shaped insertion site (FIG. 11) to chamber 1. The dissociation solution contained 200 ml collagenase and 50 mg DNase I and was dissolved in 20 ml lactated Ringer's solution. Additional lactate Ringer solution was introduced into chamber 1 to dilute the dissociation solution. The dissociation solution was then transferred to chamber 2. In order to clean the chamber 1, the lactated Ringer's solution was again introduced into the chamber 1, and the residual dissociation solution was conveyed to the chamber 2. During the tissue dissociation operation, approximately 100 ml of lactated Ringer's solution was added to chamber 2. The device was placed in an incubator at 37 degrees Celsius and frequently massaged to efficiently mix the tissue sample and dissociation solution. The tissue dissociation operation took about 45-60 minutes.

  When the clamp 1 is opened after dissociation, the released cells can enter the residue removal chamber (chamber 4 in FIG. 3A). When the clamp 2 is subsequently opened, the sample can pass through the mesh 3. Tissue debris and adipocytes were substantially removed during this procedure.

  Next, the solution flowing out of the chamber 4 was supplied to the microfluidic device 1100 under gravity. The microfluidic device 1100 is shown in FIG. 17 and is disclosed in International Application PCT / US10 / 061866, the entirety of International Application PCT / US10 / 061866 incorporated herein by reference for all purposes. The The microfluidic device included about 110 modules disposed on the flat surface of the substrate. Each module contained approximately 900 struts arranged in 4 rows. The depth of the microfluidic channel was about 30 μm.

  The output of the microfluidic device is shown in FIGS. 18A and 18B. Enriched non-adipocytes were collected substantially in the nucleated cell product fraction (FIG. 18A). The volume of the nucleated cell product fraction was reduced to about 1/8 of the input. The cells were then stained with acridine orange (2 mg / l) and imaged using a fluorescence microscope. The image shows that adipocytes are substantially absent in the product output of the microfluidic device and that non-adipose nucleated cells are substantially collected in the nucleated cell fraction. The image also shows that the cells are in good shape and that the product output is substantially free of damaged cells.

Cells isolated using the disclosed methods and apparatus were further characterized for total nucleated cell counts and adherent viable cell counts using the ADAM MC automated mammalian cell counter. The output of chamber 4 had about 6.0 × 10 ⁵ total nucleated cells per ml of treated aspirated tissue. Alpha-MEM supplemented with 10% fetal bovine serum (FBS) was used as a culture medium for adherent viable nucleated cell counts. The output of chamber 4 was sampled and cultured for 3 days at 37 degrees Celsius in a culture medium in a cell culture chamber (such as a cell culture dish or cell culture flask). After 3 days, the culture medium was discarded and the cell culture chamber was washed with Dulbecco's phosphate buffer saline solution. The 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 approximately 1.5 × 10 ⁵ per ml of aspirated treated adipose tissue.

Example 2 Method for Separating Non-Adipose Cells from Human Aspirated Adipose Tissue Using Sample Processing Bag Device Human aspirated adipose tissue was processed using the sample processing bag device shown in FIG. The apparatus includes a sample dissociation chamber (chamber 1), a waste chamber (chamber 2), and a cell purification chamber (chamber 3). The device is about 24 cm wide and about 36 cm long and is made from two polyvinyl chloride (PVC) sheets about 0.3 mm thick. The sample dissociation chamber and the cell purification chamber include a first nylon mesh (Mesh 1) and a second nylon mesh (Mesh 2). The pore size of the first mesh is about 125 μm, and the pore size of the second mesh is about 25 μm. The pore size of the first mesh is alternatively in the range of about 70 μm to about 160 μm (eg, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, or about 160 μm). The pore size of the second mesh may alternatively be in the range of about 20 μm to 50 μm (eg, about 20 μm, about 22 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, or about 50 μm).

  Human aspirated adipose tissue was collected from an approved donor using tumenic liposuction and processed within 6 hours of collection. Samples were shipped and stored at 4 degrees Celsius prior to processing.

  Initially, the stopcock 1 was set at a position where the chamber 1 and the chamber 2 were connected. Dissociation solution containing 100 mg collagenase, 100 mg hyaluronidase, and 20,000 units deoxyribonuclease was loaded into syringe 2. The sample processing bag device was connected to a bag of washing solution containing lactated Ringer's injection using a spike.

  Approximately 75 ml of aspirated adipose tissue sample was injected from port 1 into chamber 1 using a syringe with a catheter tip.

  A washing procedure was applied to wash the aspirated adipose tissue sample. To initiate the cleaning cycle, stopcock 1 was set to a closed position that fluidly disconnected chamber 1 from chamber 2 and chamber 3. About 100 ml of lactated Ringer's injection was injected into chamber 1 using syringe 1 and stopcock 2 as flow control devices. The injection is performed in the following order: (a) switching the stopcock 2 so that the cleaning solution is connected to the syringe 1, (b) drawing the cleaning solution into the syringe 1, and (c) the syringe 1 Switching the stopcock 2 to be connected to the chamber 1 and (d) injecting the cleaning solution from the syringe 1 into the chamber 1. This sequence can be repeated until the desired volume of solution is added to the dissociation chamber.

  Next, the stopcock 1 was switched so that the chamber 1 was massaged to mix the cleaning solution and the sample, and excess fluid (ie waste solution) was discharged into the chamber 2. After discharge, the first disposal cycle was completed. The wash cycle was repeated twice.

  The washing operation can include one or more washing cycles (eg, 1 cycle, 2 cycles, 3 cycles, 4 cycles, or 5 cycles).

  After washing, dissociation solution was added to chamber 1. About 100 ml of wash solution was also added to chamber 1. The sample processing bag apparatus was then incubated at 37 degrees Celsius for approximately 60 minutes incubation time. Chamber 1 was massaged during this time to mix the sample and dissociation solution. In some embodiments, other incubation times (eg, 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) can also be used. .

  After incubation, the individual cells released from the sample passed through the stopcock 1 and into the chamber 3. On the other hand, large tissue residuals were captured by the mesh 1 and left in the chamber 1. Mesh 2 in chamber 3 further removed large adipocytes and debris from the dissociated sample. Non-adipocytes, including pericytes, adipose-derived stem cells, and progenitor cells were then collected at the outflow port of chamber 3.

  In order to concentrate the released cells, removed residual red blood cells, and residues, the released cells collected at the outflow port of chamber 3 of the sample processing bag device are then transferred to WO 2011/079217 (A1 ) Passed through the first microfluidic device disclosed in The microfluidic device included 73 modules arranged on the surface of the substrate. Each module included approximately 1,300 struts organized into four rows. The microfluidic device included channels of substantially the same depth (ranging from about 35 μm to about 50 μm). In other embodiments, the microfluidic device has a channel having a depth in the range of about 30 μm to about 80 μm (eg, 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). Can be included. The cells at the outlet of the microfluidic device were concentrated to about 1/3. In other embodiments of the invention, the microfluidic device reduces the cells to less than about 1 / 2.5 (eg, about 1/3, about 1/4, about 1/5, about 1/6, about 1 / 2.5). 8, about 1/10, about 1/12, about 1/15, about 1/20, about 1/25, about 1/30, about 1/40, about 1/50, about 1/60, about 1 / 80, about 1/100, or about 1/125).

  To remove the enzyme, the cells were processed through a second microfluidic device disclosed in WO 2011/079217 (A1). The second microfluidic device included 83 modules arranged on the surface of the substrate. Each module contained approximately 900 struts arranged in 4 rows. A wash solution stream was introduced into each module, and the cells were separated from the enzyme and transported to the wash solution.

  An enzyme-linked immunosorbent assay (Elisa) was applied to measure the residual amount of collagenase after the second microfluidic device. The results show that the collagenase concentration was reduced to 1/1000 and the purified cells contained less than 0.001 mg / ml collagenase.

  Although several aspects of at least one embodiment have been described, it should be understood that various changes, 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 this disclosure. Accordingly, the above description and drawings are intended to be illustrative.

According to one aspect of the present disclosure, an apparatus for separating non-adipocytes from an adipose tissue sample is provided. The apparatus comprises a first sheet material, a second sheet material bonded to the first sheet material, and a plurality of chambers defined between the first sheet material and the second sheet material. 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 purification including an inlet and outlet in fluid communication with the sample dissociation chamber A plurality of chambers including a chamber.
In a preferred embodiment, the present invention provides the following items, for example.
(Item 1)
An apparatus for separating non-adipocytes from an adipose tissue sample,
The material of the first sheet;
A second sheet material bonded to the first sheet material;
A plurality of chambers defined between the material of the first sheet and the material of the second sheet,
A sample dissociation chamber comprising 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 purification chamber comprising an inlet in fluid communication with the sample dissociation chamber and an outlet
A plurality of chambers including
A device for separating non-adipocytes from an adipose tissue sample.
(Item 2)
The apparatus of item 1, wherein the sample dissociation chamber further comprises a mesh filter comprising pores having pore sizes ranging from 70 μm to 300 μm.
(Item 3)
The apparatus of item 1, further comprising a mesh filter contained in the cell purification chamber comprising pores having a pore size ranging from 20 μm to 50 μm.
(Item 4)
The sample dissociation chamber further includes a first mesh filter including pores having a first pore size, and the cell purification chamber includes first pores having a second pore size smaller than the first pore size. The apparatus of item 1, further comprising a two-mesh filter.
(Item 5)
The apparatus of claim 1, further comprising means for controlling a fluid connection between the sample dissociation chamber, the waste collection chamber, and the cell purification chamber.
(Item 6)
6. The apparatus of item 5, wherein the means for controlling the fluid connection includes a stopcock.
(Item 7)
The apparatus of claim 1, further comprising a flow control device configured to introduce at least one of a wash solution and a dissociation solution into the sample dissociation chamber and having an outlet in fluid communication with the sample dissociation chamber.
(Item 8)
The apparatus of claim 1, further comprising means for applying pressure to one of the sample dissociation chamber and the cell purification chamber.
(Item 9)
A downstream processing device in fluid communication with the outlet of the cell purification chamber, wherein the fluid output from the cell purification chamber is a first solution having a first concentration of one or more target cells; and And further comprising a downstream processing device comprising at least one microfluidic device configured to sort into a second solution having a concentration of the one or more target cells that is less than the concentration of the first solution, Item 2. The device according to Item 1, wherein the subject cell comprises a non-adipocyte separated from a fat tissue sample.
(Item 10)
A sterilized and substantially separated adipose tissue processing system comprising:
A tissue processing chamber comprising 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 contained in the same housing as the tissue processing chamber, the waste collection chamber including an inlet in fluid communication with the outlet of the tissue processing chamber;
A residue removal chamber including a residue removal mechanism and one of a sample collection chamber contained in the same housing as the tissue processing chamber and in fluid communication with the tissue processing chamber;
Including the system.
(Item 11)
A method of processing an adipose tissue sample in a tissue processing system comprising:
Introducing an adipose tissue sample to be processed into the first chamber through an inlet port of the first chamber;
Processing the adipose tissue sample in the first chamber;
Transporting cells from the first chamber through the outlet of the first chamber to a second chamber contained in the same housing as the first chamber through the inlet of the second chamber;
Including the method.
(Item 12)
12. The method of item 11, wherein processing the adipose tissue sample comprises dissociating the adipose tissue sample.
(Item 13)
The method of claim 11, wherein processing the adipose tissue sample includes removing excess fluid from the adipose tissue sample in the first chamber.
(Item 14)
12. The method of item 11, wherein processing the adipose tissue sample comprises rinsing the adipose tissue sample with a wash solution in the first chamber.
(Item 15)
Processing the adipose tissue sample includes washing the adipose tissue sample in the first chamber using a wash solution and disposing the adipose tissue sample in the first chamber using a dissociation solution containing at least one enzyme. 12. The method according to item 11, comprising dissociating.
(Item 16)
16. A method according to item 15, wherein the dissociation solution contains collagenase.
(Item 17)
16. The method of item 15, wherein the dissociation solution comprises collagenase, deoxyribonuclease, and hyaluronidase.
(Item 18)
16. The method of item 15, wherein dissociating the adipose tissue sample with the dissociation solution is performed at about 37 degrees Celsius.
(Item 19)
12. The method according to item 11, further comprising removing residue using a mesh filter included in the second chamber.
(Item 20)
20. A method according to item 19, wherein the mesh filter has a pore size ranging from 15 micrometers to 100 micrometers.
(Item 21)
The sample is held in the first chamber, and waste fluid passes through a mesh filter included in the first chamber and a first outlet of the first chamber. Item 12. The method of item 11, further comprising conveying to three chambers through an inlet of the third chamber.
(Item 22)
12. The method of item 11, further comprising enriching for the non-adipocyte population using a microfluidic device.
(Item 23)
24. The method of item 22, wherein the non-adipocyte comprises a stem cell.
(Item 24)
12. The method of item 11, further comprising harvesting the cell from the tissue processing system.
(Item 25)
A downstream processing device that fluidly communicates the cells to the outlet of the second chamber, wherein the cells are smaller than the first solution having a first concentration of non-adipocytes and the concentration of the first solution. 12. The method of item 11, further comprising processing in a downstream processing device comprising at least one microfluidic device configured to sort into a second solution having a concentration of non-adipocytes.
(Item 26)
25. The method according to item 24, wherein the collected cells are stromal vascular cell group cells.

Claims (26)

An apparatus for separating non-adipocytes from an adipose tissue sample,
The material of the first sheet;
A second sheet material bonded to the first sheet material;
A plurality of chambers defined between the material of the first sheet and the material of the second sheet,
A sample dissociation chamber comprising 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 plurality of chambers including a cell purification chamber including an inlet in fluid communication with the sample dissociation chamber and an outlet. An apparatus for separating non-adipocytes from adipose tissue samples.   The apparatus of claim 1, wherein the sample dissociation chamber further comprises a mesh filter comprising pores having pore sizes in the range of 70 μm to 300 μm.   The apparatus of claim 1, further comprising a mesh filter contained in the cell purification chamber comprising pores having a pore size ranging from 20 μm to 50 μm.   The sample dissociation chamber further includes a first mesh filter including pores having a first pore size, and the cell purification chamber includes first pores having a second pore size smaller than the first pore size. The apparatus of claim 1, further comprising a two mesh filter.   The apparatus of claim 1, further comprising means for controlling a fluid connection between the sample dissociation chamber, the waste collection chamber, and the cell purification chamber.   The apparatus of claim 5, wherein the means for controlling the fluid connection comprises a stopcock.   The apparatus of claim 1, further comprising a flow control device configured to introduce at least one of a wash solution and a dissociation solution into the sample dissociation chamber and having an outlet in fluid communication with the sample dissociation chamber.   The apparatus of claim 1, further comprising means for applying pressure to one of the sample dissociation chamber and the cell purification chamber.   A downstream processing device in fluid communication with the outlet of the cell purification chamber, wherein the fluid output from the cell purification chamber is a first solution having a first concentration of one or more target cells; and And further comprising a downstream processing device comprising at least one microfluidic device configured to sort into a second solution having a concentration of the one or more target cells that is less than the concentration of the first solution, The device of claim 1, wherein the subject cells comprise non-adipocytes isolated from adipose tissue samples. A sterilized and substantially separated adipose tissue processing system comprising:
A tissue processing chamber comprising 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 contained in the same housing as the tissue processing chamber, the waste collection chamber including an inlet in fluid communication with the outlet of the tissue processing chamber;
A residue removal chamber including a residue removal mechanism and one of a sample collection chamber contained in the same housing as the tissue processing chamber and in fluid communication with the tissue processing chamber. A method of processing an adipose tissue sample in a tissue processing system comprising:
Introducing an adipose tissue sample to be processed into the first chamber through an inlet port of the first chamber;
Processing the adipose tissue sample in the first chamber;
Transporting cells from the first chamber through the outlet of the first chamber to a second chamber contained in the same housing as the first chamber through the inlet of the second chamber. .   The method of claim 11, wherein processing the adipose tissue sample comprises dissociating the adipose tissue sample.   The method of claim 11, wherein processing the adipose tissue sample includes removing excess fluid from the adipose tissue sample in the first chamber.   The method of claim 11, wherein processing the adipose tissue sample comprises rinsing the adipose tissue sample with a wash solution in the first chamber.   Processing the adipose tissue sample includes washing the adipose tissue sample in the first chamber using a wash solution and disposing the adipose tissue sample in the first chamber using a dissociation solution containing at least one enzyme. 12. The method of claim 11, comprising dissociating.   The method of claim 15, wherein the dissociation solution comprises collagenase.   The method of claim 15, wherein the dissociation solution comprises collagenase, deoxyribonuclease, and hyaluronidase.   16. The method of claim 15, wherein dissociating the adipose tissue sample with the dissociation solution is performed at about 37 degrees Celsius.   The method of claim 11, further comprising removing residue using a mesh filter included in the second chamber.   The method of claim 19, wherein the mesh filter has a pore size ranging from 15 micrometers to 100 micrometers.   The sample is held in the first chamber, and waste fluid passes through a mesh filter included in the first chamber and a first outlet of the first chamber. The method of claim 11, further comprising transporting to three chambers through an inlet of the third chamber.   12. The method of claim 11, further comprising enriching for the non-adipocyte population using a microfluidic device.   24. The method of claim 22, wherein the non-adipocyte comprises a stem cell.   The method of claim 11, further comprising harvesting the cell from the tissue processing system.   A downstream processing device that fluidly communicates the cells to the outlet of the second chamber, wherein the cells are smaller than the first solution having a first concentration of non-adipocytes and the concentration of the first solution. 12. The method of claim 11, further comprising processing in a downstream processing device comprising at least one microfluidic device configured to sort into a second solution having a concentration of non-adipocytes.   The method according to claim 24, wherein the collected cells are stromal vascular cell group cells.