Microfluidic biospecimen cartridge

Abstract

The invention is directed to devices and methods for culturing, maintaining, storing, shipping and / or monitoring biospecimens. In some embodiments, devices of the invention comprise a self- contained system of reagent reservoirs connected to an array of biologically isolated chambers which are, in turn, each connected to a waste reservoir. After loading specimens and assembling the device components into an operational cartridge, specimens are maintained by controlled transfer of medium to chambers and transfer of used medium to a waste reservoir while maintaining biological isolation within each chamber.

Description

MICROFLUIDIC BIOSPECIMEN CARTRIDGE

[0001] Shipment, temporary storage, culturing and monitoring of live biological specimens is subject to a multitude of safety and environmental requirements including, but not limited to, containment, thermal control, humidity control, pH control, nutrient control, and the like. For temporary' storage and short-distance shipments, packaging typically comprises single or multi-use insulative plastic containers with specimens packed in ice or with frozen gel-packs of various sorts, e.g. Goellner et al, hit. J. Life Cycle Assess., 19: 611 -619 (2014); Pitt, editor-in-chief. Best Practices, 2ndedition, International Society for Biological and Environmental Repositories (2008).

[0002] It would be highly desirable, especially for medical or clinical applications, if there were available alternative biospecimen transport systems that were, on one hand, more flexible and capable in meeting specimen requirements than currently available shipping containers, simple to use, and self-contained, and, on the other hand, light weight and inexpensive to manufacture.SUMMARY OF THE INVENTION

[0003] The invention is directed to methods, systems and cartridges, including microfluidic devices, for culturing, maintaining, shipping, storing and / or monitoring biological specimens (the latter sometimes referred to herein as “biospecimens”).

[0004] In some embodiments, the invention provides a cartridge for holding one or more live biological samples, the container comprising; (a) a first body comprising at least one reagent reservoir in fluid communication with a plurality of passages, each passage of the plurality comprising one or more passage outlets; and (b) a second body comprising; (i) a plurality chambers in a planar array, wherein each chamber comprises a chamber inlet and a chamber outlet wherein each chamber inlet is in fluid communication with a passage outlet and optionally comprises a first filter that passes liquid but not biological material and each chamber outlet comprises a second filter that passes liquid but not biological material; and (ii) a waste reservoir in fluid communication with at least one chamber outlet; wherein the first body and the second body are configured to fit together such that the passage outlets are aligned with the chamber inlets of the plurality of chambers and a fluid-tight seal is formed therebetween and such that each chamber is in fluid communication with the reagent reservoir through its first filter. In some embodiments, a chamber inlet may not require a firstfilter. For example, some embodiments may employ gravity to maintain cells in a chamber. In some embodiments, more than one type of biospecimen may be held in a single chamber. For example, mammalian cells and stromal cells both may be loaded into a chamber.

[0005] In some embodiments, the first and second bodies of cartridges of the invention each comprise one or more layers, or more usually, each comprise pluralities of layers that provide a functionality including, but not limited to, fluid passages between components (e.g. inlet and chambers), reagent reservoirs, waste reservoirs, filtration, chambers for culturing or maintaining cells, optical window for passing a beam of light along an opical pathway with minimal attenuation, and the like. In some embodiments, an optical w indow provides for a variety of optical detection methods for detecting or assessing cells in chambers, including, but not limited to imaging of cells as w ell as fluorescence and absorbance measurements.

[0006] In some embodiments, the invention provides a method of culturing living biospecimens comprising delivering at predetermined intervals predetermined volumes of reagent to each of a plurality' of chambers each in fluid communication with each of one or more reagent reservoirs, wherein each chamber comprises an inlet comprising a first filter and an outlet comprising a second filter and w herein at least one chamber contains a living biospecimen; wherein any fluids in the chambers displaced by the step of delivering enter a waste reservoir through the second filters; and w herein the first and second filters pass fluids but not biological materials.

[0007] These above-characterized aspects, as w ell as other aspects, of the present invention are exemplified in a number of illustrated implementations and applications, some of which are shown in the figures and characterized in the claims section that follows. However, the above summary' is not intended to describe each illustrated embodiment or every implementation of the present invention.Brief Description of the Drawings

[0008] Fig. 1A illustrates diagrammatically a design ofcartridges comprising sixteen chambers.

[0009] Figs. 1B-1C illustrate diagrammatically various designs of passage networks that may be used in cartridges of the invention.

[0010] Figs. 2A-2F illustrate diagrammatically features of several embodiments of chambers of a cartridge or device of the invention and how they function in association with an appliance that provides sources of heat, pressure, optical detection, and the like.

[0011] Fig. 3 illustrates an embodiment of a cartridge of the invention wherein its chambers comprise a protrusion to capture liquid sample prior to assembly.

[0012] Figs. 4A-4C illustrate diagrammatically a particular embodiment of a cartridge of the invention.

[0013] Fig. 5 diagrammatically illustrates the use of an inflatable bladder in a reagent reservoir for forcing liquid from the reservoir to the chambers while maintaining self-containment.

[0014] Fig. 6 is a blow-up view of one embodiment of a device or cartridge of the invention showing particular versions of a reagent reservoir and reagent passages.

[0015] Figs. 7A-7B diagrammatically illustrate cartridges of the invention that are constructed from layers.

[0016] Figs. 7C-7D diagrammatically illustrate optical windows in cartridges employing epiillumination and transmission illumination, respectively, for imaging cells.

[0017] Fig. 8 A illustrates a particular array of chambers and additional layers providing fluid pathways or channels for reagent delivery and for reagent removal.

[0018] Fig. 8B illustrates layers of a particular cartridge embodiment of the invention.DETAILED DESCRIPTION OF THE INVENTION

[0019] The general principles of the invention are disclosed in more detail herein particularly by way of examples, such as those shown in the drawings and described in detail. It should be understood, however, that the intention is not to limit the invention to the particular embodiments described. The invention is amenable to various modifications and alternative forms, specifics of which are shown for several embodiments. The intention is to cover all modifications, equivalents, and alternatives falling within the principles and scope of the invention. Guidance for selecting materials and components to carry out particular functions may be found in available treatises and references on scientific instrumentation including, but not limited to, Moore et al, Building Scientific Apparatus, Third Edition (Perseus Books, Cambridge, MA); Hermanson. Bioconjugate Techniques, 3rd Edition (Academic Press. 2013); and like references.

[0020] As mentioned above, the invention is directed to methods, systems and cartridges, including microfluidic devices, for culturing, maintaining, shipping, storing and / or monitoring biological specimens. In some embodiments, devices of the invention are self-contained in the sense that after loading specimens into chambers of a device such chambers are sealed with respect to further addition or removal of biological materials, but not liquids or gases. That is, in some embodiments, after closure or sealing the chambers no further biological specimens or material can beadded to a sealed chamber and no biological specimen or biological material inside a sealed chamber can escape or be removed from the sealed chamber (nondestructively). On the other hand, in some embodiments, chambers of the invention each have an inlet and an outlet comprising filter membranes that each allow the passage of gases and liquids. In some embodiments, filter membranes allow the passage of gases and liquids but after welling passage of gases is inhibited.

[0021] In some embodiments, a device of the invention comprises a container or cartridge for holding one or more live biological samples, the container comprising the following elements: a) a first body comprising at least one reagent reservoir comprising at least one reservoir outlet in fluid communication with a plurality of passages, each passage of the plurality comprising one or more passage outlets; and b) a second body comprising: (i) a plurality chambers in a planar array, wherein each chamber comprises a chamber inlet and a chamber outlet wherein each chamber inlet is in fluid communication with a passage outlet and optionally! comprises a first filter that passes liquid but not biological materials and each chamber outlet comprises a second filter that passes liquid but not biological materials; and (ii) a waste reservoir comprising at least one waste inlet for each chamber such that each waste inlet is in fluid communication with at least one chamber outlet; wherein the first body and the second body are configured to fit together such that the passage outlets are aligned w ith the chamber inlets of the plurality of chambers and a fluid-tight seal is formed therebetween and such that each chamber is in fluid communication with the reagent reservoir through its first filter.

[0022] In some embodiments, each chamber is in fluid communication w ith the waste reservoir through its second filter. In some embodiments, a valve (e.g. 105 in Fig. IA) is disposed between a plurality of passages ((104) or passage network) to prevent backflow of liquids or air into reagent reservoir (106) in case of pressure loss or variation. In some embodiments, biological material comprises tissues, cells, bacteria, fungi, viruses, or other materials capable of replication or infection. In other embodiments, filters of the inlets and outlets of chambers may be selected for special purposes, such as, growing, shipping, storing and / or monitoring only mammalian cells, or only protozoans, in which case, for example, average pore size of first and second filters may have ranges with higher minimum pore sizes than filters used with, for example, bacterial cells. In some embodiments, first and second filters comprise the same filter material with the same filter characteristics, e.g. thickness, porosity, average pore diameter, and the like. In some embodiments, first and second filters have pore characteristics for retaining mammalian cells in a chamber. In some embodiments, first and second filters have pore characteristics for retaining bacterial cells in a chamber. In someembodiments, first and second filters have pore characteristics for retaining fungal cells in a chamber. In some embodiments, inlets do not include a first filter. In some embodiments, cells in chambers without first filters are retained in each of such chambers by fluid flow from its inlet through the chamber and through its second filter.

[0023] Cartridges of the invention allow loading, sealing of biological samples, automatic perfusion, and monitoring of the specimen growth in field locations or in a laboratory' without the requirement of specially trained engineers or specialized equipment. Once closed or operably assembled, the unit is self-contained, minimizing chance of contamination and enabling safe handling of a wide variety' of biological specimens, for example, in a microgravity environment. In some embodiments, the specimens or samples studied with this system can be cells, such as bacteria, human cell lines, fungi, plant cells, spores, and organisms, such as C. elegans worms. Organisms within a closed or operably assembled cartridge may be monitored optically (e.g. light scatter, fluorescence, or the like), electronically (e.g. resistive detection), or by other non-invasive means.

[0024] In some embodiments, thermally bonded Poly Carbonate Track Etched (PCTE) membranes on top and bottom of the chamber wells (e.g. 200 mn pore size) ensure that the cells in each of the wells are isolated from each other and the outside environment, eliminating risk of cross contamination. In other embodiments, filter membranes may be bonded to first or second bodies, or subcomponents or sublayers of such bodies (for example, inside the layers of Fig. 7A), by pressure sensitive adhesives. In some embodiments, the chamber wells may be connected through built-in microfluidic channels in the first and second bodies of the cartridge. These interconnected fluidic channels allow perfusion of growth medium and buffers through all the wells, enabling parallel experiments in separate chamber wells. In some embodiments, first and second bodies of a cartridge may contain two fluidic reservoirs for growth medium and waste storage. Fluid (e.g. growth medium, wash solutions, or the like) may be moved from the reagent reservoir(s) to the chambers and to the waste reservoir using air pressure directed through (for example) 100 nm pore size Polytetrafluoroethylene (PTFE) membranes that are bonded to the inlets and outlets of the chambers. This allows air passage but prevents liquid flow-through due to the highly hydrophobic nature of the material, thereby isolating the cartridge from the instrument and operating environment. PTFE or like membranes may also be employed in bubble traps to remove bubbles or air pockets from reagents as they flow towards chambers.

[0025] Biological samples, such as cells, are loaded into the chamber wells by the user, and the first and second bodies are assembled and locked using any of a variety of latching mechanisms, pressure sensitive adhesives, or the like. The easy sample loading design allows for both dry sample and wet sample loading, and all liquids are self-contained inside the cartridge w ith minimum chance of contamination to the outside environment. The entire cartridge may then be inserted into an appliance which contains light sources, detectors, air pumps, and the like. When the experiment isturned on, positive pressure is applied to the reservoir through the cartridge inlet port, liquid in the reservoir is pushed into each of the chamber wells, and w aste is collected in the waste reservoir.

[0026] In some embodiments, users can monitor suspension cell growth over time. In some embodiments, the size of a cartridge is 1 / 3 or 1 / 2 that of the 96 well microtiter plates. Each well may be about 3 to 3.5mm in diameter and 70-100 ul volume, with well to well spacing of about 9 mm. The well spacing and unit height are the same as units used in standard well plates, thus allowing instrument compatibility.

[0027] Fig. 1 A illustrates elements of one embodiment of a device (or cartridge) of the invention. The uppermost panel is a top view showing elements interior to first body ( 102) when assembled and in operational association with second body (114). In some embodiments, reservoir ( 106) comprises a bladder (not shown) connected to a pressure source by port (108), which may include a valve under computer control. The bladder may be inflated by predetermined volumes of air to force predetermined amounts of reagent into the chambers (e.g. 110) at predetermined times by way of passages (104). In some embodiments, on-off valve (105) is disposed between passages (104) and reagent reservoir (106) to prevent backflow. In some embodiments, a bubble trap may be positioned either before or after (i.e. upstream of or downstream of) on-off valve (105 ). Prior to assembly of first body ( 102) and second body (114), specimens are added to open chambers, e.g. (1 18), after which first body (102) and second body (114) are clamped together so that each of the chambers forms a fluid-tight seal (120). The clamping and sealing elements may vary widely. In some embodiments, first and second bodies may be assembled with gaskets using screw s, snap-on fittings, clamps, or the like, e.g. U.S. patents 3540760; 4105226; 5161834; 7841629; and U.S. patent publication 20160369922. In some embodiments, assembly of first and second bodies forces upper and lower portions of individual chambers to align and be pressed together to form a fluid-tight seal. In such embodiments, upper and lower portions of chambers may comprise a pliable material, e.g. plastic, amenable to forming a leak-proof or fluid-tight seal when pressed together. In some embodiments, individual chambers may comprise individual gaskets to form a fluid-tight seal. In some embodiments, the terms “leak proof’ or “fluid-tight” means that neither liquids nor gases can pass through the seal formed. Fig. 1 A shows upper and lower portions of chambers as substantially equal in size and shape. In some embodiments, portions of chambers on the first and second bodies prior to assembly may vary in size and volume and shape. In some embodiments, chamber portions of the second body may comprise the entire chamber or a fraction of the chamber, e.g. less than half of the post assembled chamber. In some embodiments, portions of chambers on the first and second bodies prior to assembly may vary so that the second body may comprise the entire chamber or a fraction of the chamber, e.g., as small as one quarter of the postassembled chamber. In some embodiments, second body (114) comprises the entireties of each of the chambers and a fluid-tight seal is formed with their corresponding passage outlets, which fluid-tight seal may be formed using gaskets. In some embodiments, the chambers are cylindrical in shape so that they have an annular cross-section. In other embodiments, chambers can have other crosssections, such as, square or rectangular. The number of chambers in an embodiment can vary widely depending on the nature of the specimen for which it is designed. In some embodiments, devices of the invention comprise from 2 to 384 chambers; in other embodiments, such devices comprise from 4 to 96 chambers. In some such embodiments, the sizes, volumes and arrangements of chambers may match those of microwell plates comprising the same number of wells as cartridges of the invention has chambers. Chambers of a device are typically arranged in a regular planar array, e.g. for ease of manufacture or compatibility with other instruments.

[0028] Waste reservoir ( 11 1 ) is in fluid communication with the interiors of the chambers through their second filters. In some embodiments, waste reservoir (1 1 1 ) is loaded or filled with a fluid adsorbent material (112) which prevents fluids (e.g. waste fluids) expelled from a chamber from back-flowing through the second filter back into the interior of a chamber. Typically, the volume of a waste reservoir is equal to or greater than that of the reagent reservoirs. Tn some embodiments, a waste reservoir comprises at least one vent port (e.g. 140) that releases gas displaced in the reservoir by fluids discharged from the chambers, but retains the discharged fluids. For example, the vent port may comprise a PTFE filter to prevent contamination of the inside of the cartridge. Absorbent materials may comprise polymer powers such as sodium polyacrylate, lateral flow pads, e.g. Whatman CF1, CF3, CF4, CF5, CF6, CF7, and the like. In some embodiments, the volume of the waste reservoir is at least 1.25 times the volume of the reagent reservoir (or the total volume of the reagent reservoirs if there are more than one); in other embodiments, the volume of the waste reservoir is at least 1.5 times the volume of the reagent reservoir(s). In some embodiments, the waste reservoir may also contain color indicators for visual indication of the waste level or during the initial filling to indicate the chambers / wells are full. In some embodiments, indicators may be employed in chambers, e.g. as a QC method, for detecting metabolites of cells produced during growth cycle, such as certain biomarkers, including proteins, exosomes, etc

[0029] In some embodiments, first and second filters are nanopore filters and are capable of passing an optical signal. In some embodiments, nanopore filters comprise planar materials having pores with diameters in the range of from 5 nm to 10 um. In some embodiments, nanopore filters comprise planar materials having pores with diameters in the range of from 5 nm to 100 nm, or from 5 nm to 500 nm, or from 5 nm to 1 pm. In some embodiments, nanopore filters comprise planar materials having pores with diameters in the range of from 500 nm to10 μm. In some embodiments, the values of the foregoing ranges of pore diameters are expected values. In some embodiments, first and second filters may comprise track-etched membranes, e.g. Mao et al, LabChip, 9(4): 586-591 (2009); Apel, Radiation Measurement, 34: 559-566 (2001); and the like. The pore diameter size of a filter employed may depend on the nature of tire biological specimen for containment purposes. For example, mammalian cells samples may be contained by first and second filters having significantly larger pore diameters than bacteria specimens. In some embodiments, first and / or second filters may be coated with chemicals to promote cell adhesion or cell growth. In some embodiments, only a second filter is employed with the inlet of the chamber open to the inflow of reagents. In such embodiments, the flow direction and rate prevents back flow of the specimens from escaping back into the fluidic reservoir.

[0030] In some embodiments, the first and second filters may comprise the same material and same pore specifications. In other embodiments, the first and second filter may comprise different material and pore specifications, provided that such different filter materials are selected so that living or infectious parts or portions of a specimen cannot be transported through the filters.

[0031] The plurality of passages (or “passage network” or “passage manifold,” terms used synonymously), connecting one or more reagent reservoirs to the chambers may have a variety of configurations or designs. In some embodiments, a passage network may be designed for efficient fabrication, which may include the design shown in Fig. IA, where some chambers are in fluid communication with reagent reservoirs by passage pathways of different lengths. In other embodiments, a passage network may be designed so that reagents or gases are delivered to chambers substantially at the same time by providing passages having substantially equi-distance pathways from each chamber to reagent reservoirs.

[0032] Figs. I B and 1C illustrate seven designs that may be used in cartridges of the invention. Boundaries (150, 152 and 154) define regions that chamber inlets (e.g. 156, 158, 160 and 162 of design 3) are in fluid communication so that when reagent fills (for example) region (150) it passes into substantially at the same time the chamber inlets connected to the region.

[0033] In some embodiments, chambers may comprise dried reagents and / or coatings including, but not limited to, enzymes, indicator molecules (e.g. for pH monitoring, or the like), antibiotics (such as, antibiotics for susceptibility testing), coatings of materials to promote cell growth or adhesion, or the like. Exemplary coatings include, but are not limited to, glycoprotein, laminin or fibronectin mixed with fetal bovine serum, poly-lysine orcollagen. In some embodiments, different chambers of a cartridge may have different dried reagent and / or coatings.

[0034] In some embodiments, such as that of Fig. 1A, during operation a continuous column of fluid may extend from the reservoir, through the reagent passages, through the first filters, through the chambers, through the second filters, and into the waste reservoir. By the term “continuous column” in reference to fluid moving through a passage, conduit, chamber, or other fluidic structure, means that the fluid completely fills the fluidic structure, except perhaps for minor amounts of trapped bubbles, or the like. That is, there are no air gaps that separate the fluid into separate bodies. In such embodiments, the fluid column may be in at least two states: static or moving. For example, at the beginning of operation (after biological samples are deposited into chambers), there is a mixture of sample (e.g. one or more droplets containing sample) and air in the chambers, and the fluid column is static (e.g. growth medium is still confined to the reagent reservoir). In a subsequent step, a column of reagent (e.g. growth medium) is caused to flow, or move, through the passages and into the chambers, for example, to replenish exhausted medium and / or remove waste products. The column of fluid pushes the air from the passages and chambers through the second filters and into the waste reservoir. In some embodiments, the waste reservoir comprises a vent port that allows the passage of air or gas but not the passage of liquid, so that the air displaced from the chambers and passages is released from the cartridge. After the chambers are filled with reagent, the fluid column stops and returns to a static state. In some embodiments, the control of the amount and duration of pressure on the reagent reservoir controls the starting and stopping of the fluid column and its rate of movement through the passages and chambers. Such control of pressure may be under programmed computer control, where the computer implements movement of a fluid column in accordance with a predetermined schedule. In other embodiments, a computer may implement movement of a fluid column based on information obtained from monitoring culture conditions in the chambers, such as, culture pH, CO2concentration, temperature, or the like. In this embodiment, during operation of a cartridge (e.g., in conjunction with an appliance), a continuous column of reagent moves through the chambers in one or more cycles, or states, of movement and stasis. In this embodiment, when the reagent is culture medium, and when a cycle is implemented to change the chamber medium, new medium is forced into chambers through the first filters. Mixing will occur between the new medium and the old medium, but at the same time new medium will be entering the chamber and old medium and a mixture of old and new medium will be exiting the chambers through the second filters. As more and more of the old mediumis discharged from the chambers, the new medium eventually will be the predominant fluid in the chambers (and the continuous fluid column will cease movement). The timing for such a medium change depends on several factors, including volume of the chambers, flow rate of the continuous column of medium, resistance of the filters, the nature of the biospecimens, instrument pump employed, and the like. For specific embodiments, such factors are readily evaluated by one of ordinary skill to determine durations of medium movement and stasis through the chambers.

[0035] In some embodiments, more than one reagents may be employed in a cartridge of the invention. For example, a cartridge may comprise at least two reagent reservoirs wherein one of the reservoirs comprises cell growth media and the other reservoir comprises a fixing agent. In such an embodiment (for example), cells may be used in an experiment (e.g. to assess drug resistance, or the like) after which cells may be fixed for preservation and further analysis to be carried out later. Alternatively, a fixing solution may be added into the reservoir of a single-reservoir cartridge for fixing of cells at the conclusion of an experiment.

[0036] Figs. 2A-2D provide close-up views of an embodiment of a chamber of the invention. Fig. 2A shows chamber (e.g. 216 in Fig. 2B) before assembly of first body (200) with second body (203). Section (201a) of first body (200) comprises passage (202) through which reagent from a reagent reservoir passes and flows through first filter (204) during operation. Optionally, in some embodiments, first body (200) comprises a portion of chamber wall (205). Section (201b) of second body (203) comprises second filter (208) and a portion of chamber wall (211) which, when first and second bodies (200 and 203) are separated, can be loaded with a specimen or sample (206) which may comprise liquid, cells or organisms (207) in suspension, adherent cells, tissues or the like. Section (201b) further comprises waste reservoir (209) which may further comprise absorbent material (112). Fig. 2B illustrates sealed chamber (216) formed by aligning and clamping or pressing first and second bodies (200 and 203) together so that fluid-tight seals (217) are formed between chamber portions (205 and 211). During operation, either under manual or computer control, typically a predetermined amount of reagent (214) flows through passage (202) and into interior of the chamber through first filter (204). In some embodiments, a predetermined amount of reagent (214) may be delivered to chamber interior (219) and any pre-existing or depleted reagents and newly delivered reagent (214) may mix and pass through second filter (208) so that a predetermined amount of mixed reagent remains in chamber (216). Reagent forced out of interior (219) of chamber (216) enters waste reservoir (209) and is absorbed by absorbent material (212). As illustrated in Figs. 2C and 2D, in some embodiments, first and second filters (230 and 236) and passage walls (234 and 236) comprise translucent or transparent materials so that optically based measurements can be made on specimens to assess conditions such as pH, CCF content, and the like. In some embodiments, such opticallybased measurements may be made by projecting light beam (231) from light source (226) through chamber interior (219) to detector (228) so that (for example) absorption or turbidity measures can be made. In other embodimen ts, different configurations of light sources and detectors may be present for other optically based measurements, such as fluorescence or colorimetric measurements. In either case, light sources and detectors may be mounted in an appliance (interior walls of which are shown as (224 and 225 in Fig. 2C)). One or more canridges, such as cartridge (222). are inserted into the appliance (220) so that light sources, detectors and the like are aligned with chambers, pressure sources, reagent or vent ports, heating elements, or the like.

[0037] Fig. 2E illustrates an embodiment wherein a continuous column of fluid passes through chambers. In a flow-stasis cycle, reagent (214) moves through passage (202), through first filter (204), through chamber interior (221), through second filter (208), and into waste reservoir (209) (as shown in the top panel (“Fluid in movement”) of Fig. 2E), after which reagent (214) stops movement, except for absorption (213) of reagent into absorbent material (212)(as shown in the bottom panel of Fig. 2E, “Fluid static”). In some embodiments, waste reservoir comprise a vent port (not shown) so that air in waste reservoir (209) may be released. In some embodiments, reagent in waste reservoir (209) may be absorbed by' capillary' action of absorbent material (212) and air displaced by such absorption may replace the absorbed liquid in portions of waste reservoirs (209), such as the spaces below the second filters. This has the advantage of reducing the possibility that used reagents in the waste reservoir will backflow through the second filters into the chambers. As illustrated in Fig. 2F, in some embodiments, waste reservoir (209) does not comprise a vent port, as its volume is relatively large compared with the total volume of reagent that passes through tire chambers. Thus, reagent entering the space below the second filters of waste reservoir (209) is absorbed (250, solid arrows) by' absorbent material (212) and air (or other gas) in the absorbent material replaces (252, dashed arrows) the absorbed reagent, so that the outside surface of the second filters is not directly exposed to used reagent.

[0038] In some embodiments, as illustrated in Fig. 3, it is desirable to prevent contact of a sample or specimen deposited into a chamber, particularly' a liquid sample, and the second filter prior to assembly of first (361) and second (363) bodies and delivery of reagent to the assembled chambers. Some nanoporous membrane filters have increased resistance to the passage of gases after being wetted, e.g. by' a liquid specimen. To ensure that this property does not inhibit the removal of air prior to delivery of reagent, chambers ( 360) may be fabricated to comprise protrusion or barrier (362), such as, an annular shaped ledge, scaffold, screen, or the like, that precludes deposited droplet (364) of sample before assembly (368) from contacting second filter (366). Such protrusion will prevent or inhibit a liquid sample from contacting the second filter until reagent passes through the first filter and combines with the sample. After assembly (368) and delivery of reagent, sample droplet (364)coalesces with reagent (314) so that cells of a sample may move throughout the chamber, including adhering to the top surface of second filter (366).

[0039] Figs. 4 A shows one side of a particular design of an embodiment comprising 16 chambers. First body (400) may be molded plastic and reagent passages (402) may be fabricated in a plastic sheet to form a manifold using conventional microfluidic techniques. Passages (402) may be connected to a reagent reservoir (not shown) by port (404). Fig. 4B shows the opposite side (406) of first body (400) along with second body (408). First body (400) comprises reagent reservoir (405) (which in operation further comprises a cap (not shown)) and sheet (415) comprising first filters. Second body (408) comprises array (410) of chambers, sheet (412) comprising second filters, gasket (414), and cap (416). Fig. 4C is a cut-away view of the first and second bodies (400 and 408, respectively, of Figs. 4A and 4B). Passage manifold (402) and cap (416) are shown assembled with first body (420). Port (420) allows predetermined volumes of air to enter reagent reservoir (405) to force equivalent volumes of reagent to move from reagent reservoir (405) to chambers, e.g. (434). In some embodiments, a bladder (not shown) is disposed in reagent reservoir (405) so that air injected through port (430) does not come into contact with reagent held in tire reservoir. Gasket (414) seated in a groove in body (420) forms a fluid-tight seal when cap (416) is attached. In this embodiment, second body (422) comprises the entirely of each of the chambers (e.g. 434) and fluid-tight seals are formed upon assembly by press fitting the ends of the chambers into aligned recessed regions (e.g. 432) in first body (420).

[0040] Fig. 5 is a cross-sectional view of assembled first body (502) and second body (510). In this embodiment, chambers (514) form fluid-tight seals by press fitting protruding members of passage manifold (508) into inlets of the chambers. Outlets of the chambers are in fluid communication through second filters (not shown) with waste reservoir (512). In some embodiments, one or more valves (e.g. 504) may be disposed among the fluid passages connecting the chambers (514) to reagent reservoir (505), which in this embodiment is shown with bladder (506) connected to port (516). Valves (504) may be employed to deliver reagent to different sets of chambers at different times.

[0041] Fig. 6 is a blow-up view of first body (602) showing passage manifold (604), bladder (608), gasket (610) and cap (606).

[0042] Fig. 7A shows diagrammatically a particular embodiment of a cartridge wherein the first and second bodies each comprise pluralities of layers, each including a single filter layer. (Thus, the assembled cartridge in the figure comprises two filter layers. As will be illustrated below, the size, geometry and compositions of layers employed in cartridges may vary widely. Typically the size and geometry of a layer is selected for convenient use in a laboratory, medical or research setting and for convenient and efficient fabrication. In someembodiments, layers have a card-iike, or rectilinear, shape, typically having two large dimensions and one small dimension. For example, in some embodiments, the width, length and depth of a layer may be ranges of width from 1 cm to 20 cm, length from 1 to 20 cm, and depth from 0.01 mm to 3 cm. Layers may comprise a wide variety of materials including, but not limited to, plastics, thermoplastics, silicon, glass, paper, and the like, e.g. Ren et al, Accounts, 46( 11): 2396-2406 (2013). In some embodiments, layers comprise commercially available plastic sheets that have been cut, or otherwise worked, to create structures, e.g. holes, channels, reservoirs or the like, to provide functionality. In some embodiments, a layer may comprise a sheet of filter material. In some embodiments, a layer may comprise a molded body comprising three-dimensional structures such as wells, cylinders, passages, or the like, for example, that may form reservoirs, passages or other functional components, in an assembled cartridge. The layers of Fig. 7A are illustrated as having the same size and shape only for convenience of illustration. Layers of cartridges may vary in thickness and size so that the final assembled cartridge may have different geometries than those illustrated in the figure. Layers of the first and second bodies (e.g. 732) may be separately fabricated then assembled using conventional alignment and bonding methods. Typically such boding methods are selected so that ports, chambers, passages, and the like, are sealingly fabricated without leaks and w'ith predictable fluid flow and control. In some embodiments, a layer (such as shown in Figs 7A and 7B), before assembly into first or second bodies, may itself be fabricated by the assembly of a plurality of layers (sometimes referred to herein as “sublayers”). In some embodiments, layers may comprise separate components. First body (704) may be fabricated by bonding together layers (700). Such layers may include filter layer (714), layer (712) comprising a plurality of passages, and layer (710) comprising a w all that closes passages of layer (712). In some embodiments, filter layer (714) is an end layer of first body (704) which make contact with top and end layer of second body (706). Second body (706) may be fabricated by bonding together layers (702). Such layers may comprise layer (716) comprising an array of chambers (e.g. 715) and optionally waste or reagent reservoirs, or other components, in separate regions (e.g. 730) of the layer; filter layer (718); layer (720) comprising a plurality of passages (for example, channeling fluid flow from chamber outlets to a waste reservoir); and layer (722) comprising a wall that closes passages of layer (720). The assembled and bonded layers of first body (704) and second body (706) make up (725) product (734) in the chambers of which a customer may place cells and fit together (727) to form operable cartridge (708 ) for storing or transporting the enclosed cells. Examples of layers comprising passages are shown in Fig. 8B, such as layers (852), (856) and (860).

[0043] Fig. 7B shows diagrammatically a particular embodiment of a cartridge wherein first and second bodies comprise pluralities of layers, and wherein only the first body comprises a filter layer. Thus, the assembled cartridge in the figure comprises a single filter layer; otherwise, the descriptions of the layers in the embodiment of Fig. 7 A are applicable for that of Fig. 7B. As discussed more fully below, a single filter layer may be advangeous whenever optical detection or imaging is desired that comprises directing an illumination beam perpendicular to, or across, the plane of a cartridge. Filter material of a filter layer is typically opaque or minimally transmissive, thus, minimizing the thickness and / or the number of filter layers reduces illumination beam attenuation. In some embodiments, the plane of a cartridge is the same as the planes of its layers, e.g. as illustrated in Fig. 7A. The plane of a layer is equivalent to the plane of its long, or larger, dimensions (e.g. width (x-axis) and length (y-axis)). First body (754) comprises layers (750) comprising filter layer (766), layer (764) comprising a plurality of passages that deliver reagent to chambers (e.g. 777) through filter layer (766) from a reservoir (not shown), layer (762) comprising a plurality of passages that deliver spent reagent (or growth media) from chamber outlets (e.g. 775) through filter layer (766) to a waste reservoir (not shown); and layer (760) which forms a wall or boundary, for example, closing passages of layer (762). Second body (756) comprises layers (752) comprising layer (768) that includes an array of chambers each with an inlet (e.g. 777) and an outlet (e.g. 775) (forming topologically a “U” shaped structure as illustrated in Fig. 8A); layer (770) comprising passages connecting each chamber with its outlet; and layer (772) that forms a wall or boundary' closing and forming passages of layer (770) upon assembly. As above, assembled first body (754) and second body (756) form product (779) in which a customer may insert cells into the chambers and sealingly fit the two components together to form operable cartridge (758). In some embodiments, a printed circuit board with electrodes may comprise a layer, where the electrodes may be used to monitor the cells and media for pH, O2, CO2, metabolites, and the like, in the chambers. In some embodiments, such electrodes may be used to detect or monitor cell to cell signaling.

[0044] Figs. 7C and 7D are cross sections of chambers and adjacent passages comprising optical windows that permit imaging of cells by either epi-illumination or transmission illuminzation, respectively. Fig. 7C illustrates an epi-illumination system, in which excitation beam delivery and optical signal collection occur through a single objective, may be used for imaging cells through an optical wnndow. Such epi-illumination systems with objective lens and associated optics (780 and 781) are well-known in the art, for example, as disclosed in references Huber et al, WO2024 / 211082; Wilson, editor, “Confocal Microscopy’’ (Academic Press, New York, 1990); and the like. The basic components of an epi-illumination system include excitation beam filtered through an excitation filter and then focused by a lens. The converged beam is directed towards a dichroic mirror, which reflects it through an objective lens onto passage (799c). in which cells are illuminated to reflect or emit optical signal (798c). Such optical signal (798c) is collected by the same objective lens (780) anddirected to the dichroic mirror, which is selected so that it transmits the light of optical signals (798c) but reflects the light of the excitation beam (797c). In some embodiments, an appliance (e.g. similar to that of Fig. 2D) associated with the cartridge may comprise optical components (780 and 781 of Fig. 7C or 792, 793 and 794d of Fig. 7D). In the embodiment of Fig. 7C, optical window (790c) comprises a transparent region of layer (789) which forms a wall (or floor) of chamber (782) and passage (799c). The cross section of Fig. 7C also illustrates the flow of media (arrows in bold, e.g. 785 and 786) through a chamber of a cartridge comprising layers. In particular, the embodiments of Figs. 7C and 7D each comprise a single filter layer, which is advantageous for optical detection and imaging since such embodiments reduce excitation beam attenuation and scattering, and in some embodiments, loss of optical signal. Layer (788) defines chambers (782) and passage (799c). Layer (787) is a filter layer. Layer (795c) defines passages in which growth media flows to chambers (e.g. 782) and passages (786c) through layer (795c) in which used media is transported to a waste reservoir through passage (794c). Layer (784) separates passages transporting growth media to chambers from passages (defined by layer (794c)) transporting used or spent media to a waste reservoir. Layer (794c) defines passages transporting used or spent media to a waste reservoir. Layer (783) forms a wall of passages defined by layer (794c).

[0045] Fig. 7D illustrates an embodiment similar to that of Fig. 7C except that excitation beam (794) from light source (794d) is transmitted through optical window (795d) of the cartridge to generate optical signals in passage (799d) which are collected by objective (792) and optical system (793). In this embodiment, regions of layers (796), (797), (788d — especially feature 795), and (789d) corresponding to optical window (795d) are selected to be transparent to, or to minimize attenuation of, excitation beam (794).

[0046] Fig. 8A illustrates an exemplary array of chambers (800) (comprising two identical banks of eight chambers each) along with at least two additional layers: one providing passages for delivering reagent to chambers and one providing passages for connecting chambers to conduits for waste removal. Body (800) comprises at least three layers: the top most layer comprises passages (806) for delivering reagent to the chambers through a filter layer (not shown) and passages (808) for removing waste from the chambers through the same filter layer. Passages (806) are connected to a reagent reservoir (not shown) by passage (804). The direction of reagent flow is shown in blow-up (802) of one chamber element of the chamber array on the left of the figure. The chamber element comprises at least three layers: filter layer (820), a layer comprising chamber (814) and outlet passage (816), and a layer comprising passage (818) connecting chamber (814) to outlet passage (816). Reagent flows (801) through filter layer (820) into chamber (814) then flows (803) through passage (818) to outlet passage (816) then flows (805) through filter layer (820) to aw aste reservoir (not shown). In the lower portion of Fig. 8A passage (810) connecting chamber (811) to outlet (813) has a different geometry' than passage (818) of blow-up (802) and includes optical window (812) as a further element. Optical window s may be employed in a cartridge of the invention in a variety of w ays for monitoring cells and / or media conditions, such as, pH, selected cell metabolites, CO?, and like quantities. Optical windows may be positioned so that a light beam may be passed through chambers (as illustrated in Fig. 2C) or so that a light beam may be passed through passages (as illustrated in Fig. 8A). Optical windows for detection and monitoring are w ell known to those skilled in the art of microfluidics, e.g. Daw son et al, Micromachines, 12: 1467 (2021); Tehranirokh et al. Biomicrofluidics, 7: 051502 (2013); Kuswandi et al, Analytica Chimica Acta, 601: 141-155 (2007); or the like. In some embodiments, an optical window is a pathway through a cartridge which light may pass to enable an optically based measurement of biological or chemical properties of contents of a cartridge. In some embodiments, an optical window may comprise absence of material, such as, for example, a hole, in a layer, or an addition of a transparent component to facilitate the unattenuated passage of light.

[0047] Fig. 8B illustrates cartridge (850) that comprises at least five layers that provide the functionality described in Fig. 8A. Layer (852) comprises passages that deliver reagent through filter layer (854) and through layer (856) to chambers contained layer (858). Layer (852) also comprises passages that direct fluid from chamber outlets, including orifices of layer (856) to a u'aste chamber (not shown). Filter layer (854) is sandwiched between layer (852) and layer (856) to restrict the flow of reagent or waste from the chambers or outlet passage to the interstitial space (855 ) of layer (858) which in this embodiment comprises a waste reservoir. Layer (860) comprises passages (e.g. 861) that connect chambers to their outlet passages (e.g. as described in blow-up (802) of Fig. 8A). Cartridges may also include other microfluidic components such as conventional bubble traps (e.g. (869) to prevent bubbles from entering fluid reservoirs or passages, for example, see: Skelly et al, LabChip, 8: 1733-1737 (2008); Johnson et al, J. Micromcch. Microcng. 19: 095011 (2009); Xi ct al. Microfluid Nanofluid (DOI 10.1007 / sI0404-010-0592-5); Liu et al, LabChip, 11: 1688 (2011); and the likeManufacture of Cartridges and Appliances

[0048] Cartridges of the invention, which may comprise numerous interconnected elements, such as first and second bodies, chambers, passages, valves, and the like, may be formed in, a wide variety of materials well-known in the micro fluidics field, such as, silicon, glass, plastic, or the like, e.g. Ren et al, Acc. Chem. Res., 46(11): 2396-2406 (2013). That is, devices of the invention may be fabricated as microfluidics devices using well-known techniques and methodologies of the micro fluidic field. Insome embodiments, first and second bodies of the invention may comprise a plastic, such as, polystyrene, polyethylenetetraphthalate glycol, polyethylene terephthalate, polymethylmethacrylate, polyvinylchloride, polycarbonate, cyclic olefin polymer and copolymer, thermo plastic elastomer or the like. Such bodies of the invention may be fabricated with or in plastic using well-known techniques including, but not limited to, hot embossing, injection molding, laser cutting, milling, etching, 3D printing, or the like. Guidance in the selection of plastics and fabrication methodologies may be found in the following references: Becker et al. Taianta, 56: 267-287 (2002); Fiorini et al, Biotechniques, 38(3): 429-446 (2005); Bjornson et al, U.S. patent 6,803,019; Soane et al, U.S. patent 6,176.962; Schaevitz et al, U.S. patent 6,908.594; Never et al, U.S. patent 6,838.156; and the like, which references are incorporated herein by reference.

[0049] An appliance used in association with a cartridge of the invention is a multi-use device that provides physical and / or chemical conditions or sources, which may be configured to be employed by a cartridge of a particular design. Appliances may provide a variety of physical, chemical and / or analytical functions, including, but not limited to, thermal sources, pressure and vacuum sources, mechanical actuators, one or more light sources, photo diodes, one or more detection stations to enable monitoring of conditions, and the like. Appliances for use with cartridges of the invention may be constructed using conventional engineering design principles and materials, e.g. as described in Moore (cited above).Definitions

[0050] “Microfluidics” device or “nanofluidics” device, used interchangeably herein, each means an integrated system for capturing, moving, mixing, dispensing or analyzing small volumes of fluid, including samples (which, in turn, may contain or comprise cellular or molecular analytes of interest), reagents, dilutants, buffers, or the like. Generally, reference to “microfluidics” and “nanofluidics” denotes different scales in the size of devices and volumes of fluids handled. In some embodiments, features of a microfluidic device have cross-sectional dimensions of less than a few hundred square micrometers and have passages, or channels, with capillary dimensions, e.g. having maximal cross-sectional dimensions of from about 1-2 mm to about 0.1 pm. In some embodiments, microfluidics devices have volume capacities in the range of from 100 pL to a few nL, e.g. 10-100 nL or in the range of from 100 pL to 1 pL. The dimensions of corresponding features, or structures, in nanofluidics devices are typically from 1 to 3 orders of magnitude less than those for microfluidics devices. One skilled in the art would know from the circumstances of aparticular application which dimensionality would be pertinent. In some embodiments, microfluidic or nanofluidic devices have one or more chambers, ports, and channels that are interconnected and in fluid communication and that are designed for carrying out one or more analytical reactions or processes, either alone or in cooperation with an appliance or instrument that provides support functions, such as sample introduction, fluid and / or reagent driving means, such as positive or negative pressure, acoustical energy, or the like, temperature control, detection systems, data collection and / or integration systems, and the like. In some embodiments, microfluidics and nanofluidics devices may further include valves, pumps, filters and specialized functional coatings on interior walls, e.g. to prevent adsorption of sample components or reactants, facilitate reagent movement by electroosmosis, or the like. Such devices may be fabricated as an integrated device in a solid substrate, which may be glass, plastic, or other solid polymeric materials, and may have a planar format for ease of detecting and monitoring sample and reagent movement, especially via optical or electrochemical methods. In some embodiments, such devices are disposable after a single use. In some embodiments, microfluidic and nanofluidic devices include devices that form and control the movement, mixing, dispensing and analysis of droplets, such as, aqueous droplets immersed in an immiscible fluid, such as a light oil. The fabrication and operation of microfluidics and nanofluidics devices are well-known in the art as exemplified by the following references that are incorporated by reference: Ramsey, U.S. patents 6,001,229; 5,858,195; 6,010,607; and 6,033,546; Soane et al, U.S. patents 5,126,022 and 6,054,034; Nelson et al, U.S. patent 6,613,525; Maher et al, U.S. patent 6,399,952; Ricco et al, International patent publication WO 02 / 24322; Bjornson et al, International patent publication WO 99 / 19717; Wilding et al, U.S. patents 5,587,128; 5,498,392; Sia et al. Electrophoresis, 24: 3563-3576 (2003); Unger et al. Science, 288: 113-116 (2000);Enzelberger et al, U.S. patent 6,960,437; Cao, “Nanostructures & Nanomaterials: Synthesis, Properties & Applications,” (Imperial College Press, London, 2004); Haeberle et al, LabChip, 7: 1094-1110 (2007); Cheng et al, Biochip Technology (CRC Press, 2001); and the like.

[0051] “Specimen,” or “biospecimen,” or “sample,” or “biological sample,” (which are used synonymously herein) means a quantity of material from a biological, environmental, medical, or patient source which comprises living or viable tissue, cells or viruses. In some embodiments,

[0030] “specimen,” or “biospecimen,” or “sample,” or “biological sample,” means a quantity of material from a biological, environmental, medical, or patient source which comprises living or viable tissue, cells or viruses, which may comprise mammaliancells, bacteria, fungi, or other eukaryotic or prokaryotic cells. In some embodiments, “specimen,” or “biospecimen,” or “sample,” or “biological means a quantity of material from a biological, environmental, medical, or patient source which comprises living or viable tissue or cells. In the latter embodiments cells may include eukaryotic or prokaryotic cells. In some embodiments, such cells comprise mammalian cells. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples, such as, waste water samples. A sample may include a specimen of synthetic origin, such as genetically engineered bacteria. Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, such as, fluids from nasal or other swabs, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may include materials taken Horn a patient including, but not limited to cultures, blood, saliva, tears, sweat, urine, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, semen, needle aspirates, and the like. Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.

Claims

What is claimed is:

1. A cartridge for holding one or more live biological samples, the container comprising: a first body comprising: at least one reagent reservoir in fluid communication with a plurality of passages, each passage of the plurality comprising one or more passage outlets; a second body comprising: a plurality of chambers in a planar array, wherein each chamber comprises a chamber inlet and a chamber outlet wherein each chamber inlet is in fluid communication with a passage outlet and comprises a first filter that passes liquid but not biological material and each chamber outlet comprises a second filter that passes liquid but not biological material; and a waste reservoir in fluid communication with at least one chamber outlet; wherein the first body and the second body are configured to fit together such that the passage outlets are aligned with the chamber inlets of the plurality of chambers and a fluid-tight seal is formed therebetween and such that each chamber is in fluid communication with the reagent reservoir through its first filter.

2. The cartridge of claim 1 wherein said first body comprises one or more layers and wherein said second body comprises one or more layers.

3. The cartridge of claim 2 wherein said first body comprises a plurality of layers and wherein said plurality of passages comprises at least one layer of said first body.

4. The cartridge of claim 3 wherein at least one of said plurality of layers comprises at least one filter layer.

5. The cartridge of claim 4 wherein said first filter of each of said chambers comprises a different non-overlapping region of said at least one filter layer whenever said first body and second body are fitted together.

6. The cartridge of claim 2 wherein said second body comprises at least one layer comprising said plurality of chambers.

7. The cartridge of claim 6 wherein said second body further comprises at least one filter layer and wherein said second filter of each of said chambers comprises a different non-overlapping region of said at least one filter layer.

8. The cartridge of claim 7 wherein said second body further comprises a plurality of passages placing each of said chamber outlets in fluid communication with said waste reservoir through said at least one filter layer of said second body.

9. The cartridge of claim 4 wherein said first filter and said second filter of each of said chambers comprises a different non-overlapping region of said at least one filter layer whenever said first body and said second body are fitted together.

10. The cartridge of claim 9 wherein said at least one filter layer is a single filter layer having a plane corresponding to its long dimensions and said cartridge comprises at least one optical window perpendicular to the plane of said filter layer.

11. The cartridge of claim 9 further comprising a plurality of passages placing each of said chamber outlets in fluid communication with said waste reservoir through said at least one filter layer of said first body.

12. The cartridge of claim 11 wherein said plurality of passages placing each of said chamber outlets in fluid communication with said waste reservoir comprises at least one layer of said first body.

13. The cartridge of claim 1 wherein said waste reservoir comprises absorbent material.

14. The cartridge of claim 1 wherein a reagent forms a continuous column of fluid from said at least one reagent reservoir through said passages and said chambers.

15. The cartridge of claim 1 wherein said plurality of passages places each of said chambers in fluid communication with each of said reagent reservoirs by an equi-distant pathway.

16. The cartridge of claim 1 wherein said reagent in at least one of said reagent reservoirs is a culture medium for living cells and wherein the culture medium comprises an optical indicator compound indicative of culture conditions.

17. The cartridge of claim 1 wherein for each chamber an optical pathw ay extends through said chamber so that a light beam can be projected along the optical pathway and signals from optical indicators detected along the optical pathway.

18. A method of culturing living biospecimens comprising: providing a continuous column of reagent that delivers reagent from at least one reagent reservoir by passages of a passage network to a plurality of chambers each comprising an inlet comprising a first filter and an outlet comprising a second filter, wherein at least one chamber comprises a living biospecimen; delivering a predetermined volume of reagent to each chamber at predetermined intervals by forcing the continuous column of reagent through the first filters; wherein any fluids in the chambers displaced by the step of delivering enter a waste reservoir through the second filters; and wherein the first and second filters pass fluids but not biological materials.

19. The method of claim 18 wherein said predetermined volume is the same for each chamber.

20. The method of claim 18 wherein said biospecimen comprises mammalian cells and said reagent is a grow medium.

21. The method of claim 18 further comprising monitoring conditions of said chambers and wherein said predetermined intervals depend on the conditions monitored in said chambers.

22. The method of claim 18 wherein said waste reservoir comprises absorbent material.

23. The method of claim 18 wherein said passages of said passage network provide equi-distant pathways between each of said chambers and each of said at least one reagent reservoir.

24. The method of claim 18 wherein each of said first filters and said second fillers comprise different non-overlapping regions of a single filter layer.

25. A method of culmring living biospecimens comprising: delivering at predetermined intervals predetermined volumes of reagent to each of a plurality of chambers each in fluid communication with each of one or more reagent reservoirs, wherein each chamber comprises an inlet comprising a first filter and an outlet comprising a second filter and wherein at least one chamber contains a living biospecimen; andwherein any fluids in the chambers displaced by the step of deli vering enter a waste reservoir through the second filters: and wherein the first and second filters pass fluids but not biological materials.

26. The method of claim 25 further comprising absorbing said displaced fluids by absorbent material disposed in said waste reservoir.

27. The method of claim 25 wherein each of said first filters and said second filters comprise different non-overlapping regions of a single filter layer.

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