Patent ID: 12221601

DETAILED DESCRIPTION OF THE INVENTION

Encapsulation Devices

Encapsulation devices are devices for holding cells or tissues. The encapsulation device (110) shown inFIG.1Ais a single-chamber encapsulation device. The device (110) comprises an inner lumen for holding the cells (102) or tissue and at least one membrane, e.g., a vascularization membrane (120), which is impermeable to cells. In some embodiments, the device (100) further comprises an immunoisolation membrane (130). Non-cell factors or molecules (150) can escape the cell impermeable membrane. The device (110) also comprises a port (180) to access the lumen for loading the cells or tissue.FIG.1Bshows a cross-sectional view of an encapsulation device. The cells are encapsulated in a lumen (114) by a two-layer membrane envelope, a vascularization membrane (120) and an immunoisolation membrane (130). The device (110) also has structural support, e.g., mesh, seals, etc. Vasculature may grow around the device (110).

In some embodiments, the encapsulation devices (110) comprise a vascularization membrane (120) and immunoisolation membrane (130). In some embodiments, the encapsulation devices (110) comprise just the vascularization membrane (120). This allows blood vessels to grow within the transplanted tissue.

In the examples shown inFIG.1AandFIG.1B, the cells therein are about 5-15 μm in diameter. The outer membrane, the vascularization membrane (120), has a pore size from 5-10 μm. The vascularization membrane (120) is about 15 μm thick. The immunoisolation membrane (130) has a pore size of about 0.4 μm. The immunoisolation membrane (130) is about 30 μm thick. In some embodiments, the membranes (120,130) are constructed from materials such as polytetraflouroethylene (PTFE) or other similar material. The present invention is not limited to the aforementioned pore sizes and thicknesses of the membranes used therein. The present invention is not limited to the aforementioned materials.

The encapsulation devices (110) may be constructed in various shapes and sizes and with various lumen volumes. For example, in some embodiments, the lumen has a volume of about 4.5 μI. In some embodiments, the lumen has a volume of 20 μI. In some embodiments, the lumen has a volume of 40 μI. In some embodiments, the device (110) is from 4 to 5 cm in length. In some embodiments, the device (110) is from 2 to 5 cm in length, e.g., 3 cm. In some embodiments, the device (110) is from 5 to 10 cm in length. The present invention is not limited to the aforementioned dimensions and lumen volumes. For example, in some embodiments, the lumen has a volume of about 100 μI. In some embodiments, the lumen has a volume of about 200 μI. In some embodiments, the lumen has a volume from 2 to 50 μI. In some embodiments, the lumen has a volume from 10 to 100 μI. In some embodiments, the lumen has a volume from 40 to 200 μI. In some embodiments, the lumen has a volume from 100 to 300 μI. In some embodiments, the lumen has a volume from 200 to 500 μI.

In some embodiments, within the encapsulation devices (110), there may be layers of cells or tissue, e.g., multiple lumens within the device (110). For example, an encapsulation device (110) may comprise two chambers or lumens. In some embodiments, the device comprises more than two chambers or lumens, e.g., 3 chambers or lumens, 4 chambers or lumens, 5 chambers or lumens, etc.FIG.2AandFIG.2Bshow examples of an encapsulation with two lumens (two chambers) that are separated by a gas channel (160).FIG.2AandFIG.2Balso show vascularizing membrane and microvasculature. The blood vessels embed into the vascularizing membrane.

In some embodiments, the chamber or lumen comprises a single layer of cells. In some embodiments, the chamber or lumen comprises two layers of cells. In some embodiments, the chamber comprises three or more layers of cells. In some embodiments, islet spheroids (about 150 μm in size) are used (shown inFIG.2A,FIG.2B). In some embodiments, a dual layer of the islet spheroids is used (lumen thickness would be about 300 μm in the chamber or in each chamber). In some embodiments, a third layer is supported depending on the metabolic activity and other characteristics of the spheroids/cells used. Note spheroids may not be touching each other in some configurations and the space between them may be 1 or 2 spheroids apart (e.g., 150 μm, 300 um), or more or less.

Methods and Systems for Monitoring Cells

FIG.3Ashows a schematic view of a system (100) comprising a bioreactor and encapsulation device with cells disposed therein. Liquid surrounds the encapsulation device (110). In some embodiments, the encapsulation device is a single chamber device. In some embodiments, the encapsulation device is a dual chamber device with a gas channel (e.g., oxygen channel) disposed between the chambers (e.g., to allow for higher density). Liquid media flows in via an inlet (106) (which may comprise a flow cell (412) with an oxygen sensor (410)) and flows out of an outlet (107) (which may comprise a flow cell (412) with an oxygen sensor (410)). In some embodiments, the sensors on the inlet and outlet are for oxygen consumption rate measurements (which may be used as a means of evaluating viability and health of the cells).

FIG.3Bshows a schematic view of a system that comprises a dual chamber device (e.g., used in a system shown inFIG.3A) having a first chamber (113a) and a second chamber (113b) separated by a gas channel (160). Gas (e.g., air, oxygen) is delivered through the middle of the chambers via the gas channel to support viability and function of the cells. The gas channel does not leak into the media of the bioreactor, and does not cause leakage of the media in the bioreactor (it is separate from the media and inlet/outlet of the bioreactor).

Insulin secretion requires physiological temperature, so the evaluation of cells in the device for insulin secretion needs to be done at 37° C. (assessments at lower temperatures are not accurate).

Oxygen consumption rate (OCR) cannot be accurately evaluated with gas flowing through the gas channel. For OCR, gas flow needs to stop, but the temperature is reduced so that there is sufficient oxygen to the cells in the center of the device and the OCR is not oxygen limited.

OCR measurements may be performed at temperatures at temperature below 37° C. (e.g., 7° C. to 27° C., e.g., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., etc.). Temperature may be adjusted to lower values as a function of metabolic activity and cell density within the devices. OCR is predictably dependent on temperature so that the health of the cells can still be estimated by appropriate temperature correction (Arrhenius type relationship). For example, OCR declines predictably with decreasing temperature in the range of 7° C. to 37° C.

FIG.4is a schematic view of a system of the present invention. A bioreactor (105) houses an encapsulation device (110), e.g., a single chamber, a dual chamber, etc., with cells. Media may be pumped via a pump through a heat or gas exchanger (or combined heat and gas exchanger), which may bring the media to a particular temperature and to a particular oxygen level. Media then flows through the inlet (106) and through an oxygen sensor (410), through the bioreactor (105) and out the outlet (107) and through the other oxygen sensor (410). Samples (for insulin secretion evaluation) may be obtained from a point outside of the outlet. The media may, for example, be recycled and flow through a water bath or incubator. Or, the media may be discarded.

In some embodiments, the combined heat and gas exchanger (or the separate heat exchanger) heats the media to a temperature between 7° C. to 37° C. (Evaluation of insulin secretion may be performed at 37° C., and then the temperature may be lowered for OCR measurements.) In some embodiments, the gas exchanger oxygenates the media to a percentage from 0-100% (e.g., 40%). Oxygen levels may be selected depending on the amount of exposure time.

For insulin secretion function measurements, high oxygen (e.g., 40% oxygen) may be present in the middle of the dual cell chamber device (along with a temperature of 37° C.).

For oxygen consumption rate (OCR) measurements, no gas is provided to the middle of the dual cell chamber. Temperature is reduced (e.g., to a temperature between 7° C. and 17° C.).

The present invention features methods and systems for monitoring cells (e.g., cell viability, oxygen consumption rates, insulin secretion levels, etc.) in an encapsulation device in real-time before transplantation (e.g., during storage) and/or after transplantation.FIG.3andFIG.4show examples of systems and devices for storing cells in encapsulation devices. These devices may be equipped with sensors (410) and readers and other features to help with real-time measurements of parameters of the cells to determine cell viability.

In some embodiments, the media is at a temperature from 4 to S° C., e.g., 4° C., 5° C., 6° C., 7° C., S° C. In some embodiments, the media is at a temperature from 2 to S° C. In some embodiments, the media is at a temperature from 4 to 10° C. In some embodiments, the media is at a temperature from 2 to 15° C. In some embodiments, the media is at a temperature from 10 to 20° C. In some embodiments, the media is at a temperature from 20 to 30° C. In some embodiments, the media is at a temperature from 30 to 3S° C. In some embodiments, the oxygen levels are around 40%. In some embodiments, the oxygen levels are less than 40% or more than 40%. In some embodiments, the oxygen level is from 0-5%. In some embodiments, the oxygen level is from 5-15%. In some embodiments, the oxygen level is from 15-25%. In some embodiments, the oxygen level is from 25-35%. In some embodiments, the oxygen level is from 35-40%. In some embodiments, the oxygen level is from 40-50%. In some embodiments, air is used (e.g., oxygen level is about 21%). The present invention is not limited to these temperatures or oxygen levels. Oxygen levels may vary as well. In some embodiments, a particular oxygen level is used initially and then the oxygen level is increased or decreased at a later time. In some embodiments, oxygen is turned on and then off. In some embodiments, oxygen is turned off and then on. In some embodiments, oxygen is turned on and off in a cycle for a period of time or indefinitely.

One or more sensors (e.g., two sensors, three sensors, four sensors, etc.) are integrated into the system, e.g., operatively connected to the encapsulation device or positioned appropriately in proximity to the encapsulation device. The sensors can help measure oxygen consumption rates, insulin secretion level, glucose levels, lactate levels, pH levels, carbon dioxide levels, or a combination thereof.

For example, in some embodiments, a first oxygen sensor is disposed at an inlet of the encapsulation device and a second oxygen sensor is disposed at an outlet of the encapsulation device, wherein the two sensors are used for oxygen consumption rate (OCR) measurements. For example, the difference in pO2 between the first oxygen sensor and the second oxygen sensor can be used to determine OCR of the encapsulated cells. This is a measure of tissue viability.

The sensors (e.g. oxygen sensors) may be used to help determine when the cells are dead (e.g., via oxygen sensors, etc.). Without wishing to limit the present invention to any theory or mechanism, cells are likely dead if there is generally no difference in oxygen levels inside and outside the device. Typically there is a difference (a gradient) in oxygen levels between the inside and outside of the device because oxygen is being consumed by live cells. Thus, no difference would be indicative of no oxygen consumption, thus the cells are likely dead. A bigger difference (gradient) in oxygen levels between the inside and outside of the device would indicate there are more viable cells. A user may determine how many cells are dying by determining the change in oxygen gradient.

The flow of oxygen gas into the encapsulation device can interfere with OCR measurements of the cells. Thus, the methods of the present invention comprise ceasing oxygen gas delivery to the cells. The methods for measuring OCR further comprise reducing the temperature of the encapsulation device and/or media surrounding the device to a temperature between 4 and 8 degrees C., which can help reduce metabolic rate of the cells and thus reduce their oxygen requirements (allowing the oxygen gas delivery to be shut off). The OCR measurements may be continuous. Without wishing to limit the present invention to any theory or mechanism, it is believed that keeping the temperature low during OCR measurements can help improve accuracy of the measurements since OCR measurements at higher temperatures can be affected by the depletion of oxygen within the packed cells due to the higher oxygen consumption be these cells at higher temperatures.

Measurements of potency of cells, e.g., measurements of glucose-stimulated insulin secretion (GSIS) may be made when the cells are at a temperature of about 37° C., or a temperature from 34 to 40° C. (e.g., 34° C., 35° C., C, 36° C., 37° C., 38° C., 39° C., 40° C.).

Insulin can be measured every minute or at shorter intervals or longer intervals as appropriate. Methods of measuring GSIS are known to one of ordinary skill in the art. Without wishing to limit the present invention to any theory or mechanism, it is believed that the flow of oxygen gas in the encapsulation device may be critical for proper GSIS measurements.

The present invention also features methods of helping to reduce cell damage (e.g., ischemic damage, physical damage, etc.) during loading of an encapsulation device. In some embodiments, the methods feature reducing the temperature of the cells, e.g., to a temperature from 4 to 8 degrees C. (which reduces the metabolic rate of cells). The cells may be in a media such as a conventional culture media such as serum supplemented or human serum albumin (HSA) supplemented RPMI, DMEM, or any other appropriate media. The methods may feature delivering oxygen gas to the device and or media. In some embodiments, pluronic acid is used during loading in order to avoid cell damage. This may be beneficial for optimization of cell loading. Also, it is possible that OCR measurements at low temperatures (e.g., from 4 to 8 degrees C.) may help determine the number of viable cells in a device during loading.

The disclosures of the following U.S. Patents are incorporated in their entirety by reference herein: PCT/US2011/055157.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the claims are exemplary and for ease of review by the patent office only, and are not limiting in any way. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting of” is met.

The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.