Devices and methods for delivering cell compositions

Abstract

Provided herein is a method of cell transplantation using an injection attachment having a hub containing a plurality of cells and a cannula extending from a distal end of the hub, the method including orienting the injection attachment so that the cannula faces downward until the plurality of cells settle together adjacent the distal end, inserting the cannula into tissue of a subject, and using the injection attachment to inject the quantity of cells into the tissue of the subject.

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of priority to U.S. Provisional Application No. 63 / 412,195, filed September 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to the field of cell therapy. More particularly, the present invention relates to devices and methods for delivering cell compositions to a subject and methods of use thereof. [Background technology]

[0003] Cell therapy involves the transfer of autologous or allogeneic cellular material to a patient for medical purposes. Cell-based therapy involves the delivery of cells to the circulatory system or tissues to treat multiple diseases. Today, cell therapy continues to evolve and is undergoing ongoing investigation into clinical safety and efficacy. Cell therapy combines stem cell-based and non-stem cell-based single-cell or multi-cell therapies. It typically uses autologous or allogeneic cells, may involve genetic engineering or manipulation in the formulation, and can be administered locally or as an injectable drug, infusion, bioscaffold, or scaffold-free system. Cell therapy spans multiple therapeutic areas, such as regenerative medicine, immunotherapy, and cancer therapy. Currently, most cell therapies are in early stages of development (Phase 1 / 2), with some exceptions that represent current best practice in specific situations (e.g., bone marrow / stem cell transplants, hepatocyte transplants, skin equivalents) or are approved for specific indications, such as PROVENGE® (sipuleucel-T), LAVIV® (azficel-T), MACI® (autologous cultured chondrocytes on porcine collagen), and KYMRIAH™ (tisagenlecleucel), among others (El-Kadiry et al., Front. Med., 8; 2021).

[0004] Pluripotent stem cells (PSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are being used to study the progression of disease processes and as potential therapeutics in multiple organ systems. In recent years, there has been growing interest in the use of PSC-based transplants to treat retinal disorders in which retinal cells are functionally damaged or lost due to degeneration. Preclinical trials in animal models of retinal disease have demonstrated improved visual outcomes after subretinal transplantation of PSC-derived photoreceptor or retinal pigment epithelial (RPE) cells. Death of retinal photoreceptor cells (PRs; rods and cones) due to inherited retinal disease (IRD) or injury is the leading cause of untreatable blindness worldwide. IRD (also known as photoreceptor degenerative disease) can manifest as either a loss of primarily rod PRs, a loss of primarily cone PRs, or a simultaneous loss of both rod and cone PRs. Once lost, PRs cannot regenerate, and treatment options for these patients are limited or nonexistent. These patients can be treated with allogeneic human induced pluripotent stem cell (iPSC)-derived photoreceptor progenitor cells (iPRP) as a cell-based PR replacement therapy for IRD.

[0005] For cell-based therapies, the successful transfer of preclinical research to clinical practice requires a means of cell delivery that is effective at the scale of human patients. There are several injection strategies for cell-based therapies. Direct injection involves the injection of cells into the body using a needle and syringe or through a delivery device such as a port or catheter / reservoir system. Intravenous injection is into the bloodstream and is the most easily accessible injection route for patients. Transplant cells are usually delivered by a guided straight cannula or needle. Generally, this cannula is connected to some form of syringe used to dispense the cell suspension.

[0006] Problems associated with cell therapy delivery include unintended deposition of cells at non-target locations, unpredictable cell dosing at the intended target site, and even substantial loss of the cell suspension. Cell delivery cannulas are typically connected to an external syringe via a Luer lock or similar coupling mechanism. This design has several drawbacks. First, most syringes dispense relatively large volumes with small plunger movements, making it difficult to manually deliver precise, small doses of cell suspension. To address this issue, incorporating a mechanical or electrical drive to control the translational movement of the plunger can be beneficial. Second, mechanical forces at the transition point between the syringe and the catheter can potentially damage the cells. The inner diameter of the syringe is typically larger than the inner diameter of the injection cannula. As a result, cells and fluids experience a significant increase in linear velocity as they pass through the syringe and enter the cannula. This creates differential velocities along the length of the cells, known as propulsion forces, which are thought to significantly contribute to cell damage during injection. Additionally, cells are also exposed to shear stress because cells and fluid in the center of the cannula move faster than cells and fluid at the outer boundary (Potts et al., Surg Neurol Inst., 4(S22-S30):2013). Summary of the Invention [Problem to be solved by the invention]

[0007] Many cell therapy approaches aim to deliver high-density single-cell suspensions to diseased or injured sites in the body. However, cell aggregates maintain viability, cell activity, and phenotype beyond that of single cells, even in non-adherent matrices, allowing for the delivery of higher cell densities with enhanced proliferation and differentiation capabilities. Thus, there is an unmet need for improved methods and devices for cell therapy, particularly for the delivery of cell aggregates and for the delivery of precise, high-density doses to target tissues, without cell damage or loss. [Means for solving the problem]

[0008] In a first embodiment, the present disclosure provides a method of cell transplantation comprising injecting cells into tissue of a subject using a cell delivery device comprising a cannula having a funnel-shaped hub.

[0009] In certain aspects, the present hubs are essentially free of catch points that can increase shear or turbulence as cells move through the hub. In certain aspects, the hubs are generally smooth as cells pass through the hub and into the cannula shaft. In certain aspects, reduced turbulence or shear helps improve cell viability, while catch points cause cell attrition and damage. In certain aspects, the present hubs are essentially free of catch points and are largely smooth compared to hubs without funnel shapes, allowing for increased release of cellular material (i.e., less material is retained within the hub).

[0010] In certain aspects, the cannula hub is less than 30 mm in length. In some aspects, the cannula comprises a cannula tip of 30 gauge or less. In certain aspects, the cannula tip is blunt. In other aspects, the cannula tip is sharp. In some aspects, the cannula tip is further defined as a needle tip. In some aspects, the cannula tip is a 34, 33, 32, 31, or 30 gauge cannula tip. In some aspects, the cannula tip is a 33 gauge cannula tip. In certain aspects, cells are loaded into a cannula having a cannula tip of 30 gauge or less. In some aspects, cells are loaded into a cannula having a 33 gauge cannula tip. In some aspects, cells are loaded into a cannula and injected into the tissue of the subject through the tip of the same cannula. In certain embodiments, the cannula tip is not altered between loading the cells into the cannula and injecting the cells into the tissue of the subject (eg, the eye).

[0011] In some aspects, the cannula tip is fabricated from a flexible polyimide material or metal. In some aspects, the cannula tip is flexible. In certain aspects, the cannula tip is rigid.

[0012] In certain aspects, the hub includes a one-way check valve. In certain aspects, the hub includes a coupling mechanism. In some aspects, the coupling mechanism is a luer lock. In certain aspects, the luer lock is coupled to a dosing mechanism. Controlled pressure can be applied to the dosing mechanism to deliver the dose. In some aspects, the dosing mechanism is a syringe. In some aspects, the syringe is a microinjection syringe. In certain aspects, the syringe is further coupled to tubing. In some aspects, the tubing is coupled to a pressure control system. In certain aspects, the cells are delivered from the cell delivery device at a controlled pressure. In some aspects, the cells are not redistributed or resuspended before loading into the cell delivery device. In some aspects, the cells are redistributed or resuspended before loading into the cell delivery device. In certain aspects, the cells are redistributed or resuspended by vortexing or manual agitation.

[0013] In some aspects, the cells are injected into the eye of the subject. In certain aspects, the cells are injected subretinal. In some aspects, the cells are further defined as cell aggregates. In certain aspects, the cells are further defined as single cells. In some aspects, the cells are in a formulation buffer. In some aspects, the formulation buffer is a balanced salt solution. In certain aspects, the balanced salt solution further comprises benzonase and / or human serum albumin. In some aspects, the cell aggregates are not in suspension when injected into the tissue of the subject. In certain aspects, the cells reside within the cannula for at least 1 minute (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, e.g., 1-3, 2-4, 3-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 9-12, 10-13, 11-14, 12-15, 13-16, 15-17, 16-18, 17-19, 18-20, 1-5, 5-10, 10-15, or 15-20 minutes) between loading and injection to allow for aggregate sedimentation within the cannula hub. In certain embodiments, the cells reside within the cannula for at least 1 minute (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, e.g., 1-3, 2-4, 3-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 9-12, 10-13, 11-14, 12-15, 13-16, 15-17, 16-18, 17-19, 18-20, 1-5, 5-10, 10-15, or 15-20 minutes) between loading and injection to allow for settling of the cells, e.g., single cells, within the cannula hub. In certain embodiments, the cannula is oriented downward to allow for settling of the cells, e.g., single cells, within the cannula hub.

[0014] In certain embodiments, the cell aggregates are photoreceptor progenitor cell aggregates. In certain embodiments, the cells are retinal progenitor cells (RPE) and / or photoreceptor progenitor cells. In some embodiments, the cell aggregates are RPE and / or photoreceptor progenitor cells. In some embodiments, the cells are injected in a volume of less than 200 μL. In some embodiments, the cells are injected in a volume of less than 100 μL. In some embodiments, the cells are injected in a volume of about 50 μL (e.g., about 25-50 μL, 50-75 μL, 75-100 μL, 100-150 μL, or 150-200 μL, e.g., about 25, 50, 75, 100, 125, 150, 175, or 200 μL). In some embodiments, at least 100,000, 250,000, 500,000, or 1 million cells are injected. In certain embodiments, at least 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, or 15 million cells are injected. In some embodiments, at least 25% of the cells loaded into the cell delivery device are injected into the subject's tissue. In some embodiments, at least 30% (e.g., 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the cells loaded into the cell delivery device are injected into the subject's tissue.

[0015] Further embodiments provide methods of treating an ocular condition in a subject, comprising transplanting cells into the subject's eye using a cell delivery device comprising a cannula having a funnel-shaped hub. In certain aspects, the cells are injected subretinal. In some aspects, the cannula hub is less than 30 mm in length. In certain aspects, the cannula comprises a 30-gauge or smaller cannula tip. In some aspects, the cannula tip is blunt. In certain aspects, the cannula tip is sharp. In some aspects, the cannula tip is further defined as a needle tip. In certain aspects, the cannula tip is a 34-, 33-, 32-, 31-, or 30-gauge cannula tip. In certain aspects, the cannula tip is a 33-gauge cannula tip. In some aspects, the cells are loaded into a cannula having a 30-gauge or smaller cannula tip. In certain embodiments, the cells are loaded into a cannula having a 33 gauge cannula tip.

[0016] In some aspects, cells are loaded into a cannula and injected into the subject's eye through the tip of the same cannula. In some aspects, the cannula tip is not changed between loading into the cannula and injecting into the subject's eye. In some aspects, the cannula tip is fabricated from a flexible polyimide material or metal. In some aspects, the cannula tip is flexible. In certain aspects, the cannula tip is rigid.

[0017] In certain aspects, the hub includes a one-way check valve. In some aspects, the hub includes a coupling mechanism. In some aspects, the coupling mechanism is a luer lock. In certain aspects, the luer lock is coupled to an administration mechanism. In some aspects, the administration mechanism is a syringe. In some aspects, the syringe is a microinjection syringe. In certain aspects, the syringe is further coupled to tubing. In some aspects, the tubing is coupled to a pressure control system.

[0018] In certain aspects, the cells are delivered from the cell delivery device at controlled pressure. In some aspects, the cells are not redistributed or resuspended before loading into the cell delivery device. In certain aspects, the cells are redistributed or resuspended before loading into the cell delivery device. In some aspects, the cells are redistributed or resuspended by vortexing or manual agitation. In some aspects, the cells are injected subretinal.

[0019] In certain aspects, the cells are further defined as cell aggregates. In some aspects, the cells are further defined as single cells. In some aspects, the cells are in a formulation buffer. In certain aspects, the formulation buffer is a balanced salt solution. In some aspects, the balanced salt solution further comprises benzonase and / or human serum albumin. In certain aspects, the cell aggregates are not in suspension when injected into the tissue of said subject. In some embodiments, the cells reside within the cannula for at least 1 minute (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, e.g., 1-3, 2-4, 3-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 9-12, 10-13, 11-14, 12-15, 13-16, 15-17, 16-18, 17-19, 18-20, 1-5, 5-10, 10-15, or 15-20 minutes) between loading and injection to allow for settling of the cells (e.g., aggregates) within the cannula hub. In certain embodiments, the cannula is pointed downward to allow for settling of the cells (e.g., aggregates) within the cannula hub. In some embodiments, the cell aggregates are photoreceptor progenitor cell aggregates. In particular embodiments, the cells are retinal progenitor cells (RPE) and / or photoreceptor progenitor cells. In some embodiments, the cell aggregates are RPE and / or photoreceptor progenitor cells. In particular embodiments, the cells are injected in a volume of less than 200 μL. In some embodiments, the cells are injected in a volume of less than 100 μL. In some embodiments, the cells are injected in a volume of about 50 μL (e.g., about 25-50 μL, 50-75 μL, 75-100 μL, 100-150 μL, or 150-200 μL, e.g., about 25, 50, 75, 100, 125, 150, 175, or 200 μL). In particular embodiments, at least 100,000, 250,000, 500,000, or 1 million cells are injected. In certain embodiments, at least 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, or 15 million cells are injected, hi some embodiments, at least 25% of the cells loaded into the cell delivery device are injected into the subject's eye.In some aspects, the ocular condition is an injury, an injury caused by a genetic retinal disease, age-related macular degeneration (AMD), hereditary macular degeneration, Stargardt's macular dystrophy, Best's disease, choroideremia, diabetic retinopathy, retinal vascular disease, retinopathy of prematurity (ROP), or a viral infection of the eye. Injury to the eye can include a contusion, puncture, scratch, penetrating injury, perforating injury, or injury caused by an intraocular foreign body.

[0020] Further embodiments provide a cannula device for cell transfer, comprising a tubular hub, the tubular hub comprising an outer wall, an inner wall, a first end, and a second end, the first end having a tapered funnel shape toward the second end. In some aspects, the inner diameter of the tubular hub decreases as measured from the first end toward the second end. In certain aspects, the hub is essentially free of catch points. In some aspects, the hub has a smooth surface.

[0021] In certain aspects, the present hubs are essentially free of catch points that can increase shear or turbulence as cells move through the hub. In certain aspects, the hubs are generally smooth as cells pass through the hub and into the cannula shaft. In certain aspects, reduced turbulence or shear helps improve cell viability, while catch points cause cell attrition and damage. In certain aspects, the present hubs are essentially free of catch points and are largely smooth compared to hubs without funnel shapes, allowing for increased release of cellular material (i.e., less material is retained within the hub).

[0022] In some embodiments, the first end is attached to a cannula tip. In certain embodiments, the cannula tip is blunt. In some embodiments, the cannula tip is sharp. In some embodiments, the cannula tip is further defined as a needle tip.

[0023] In some embodiments, the tubular hub has a length of less than 30 mm and the cannula tip is 30 gauge or smaller. In certain embodiments, the cannula tip is a 34, 33, 32, 31, or 30 gauge cannula tip. In some embodiments, the cannula tip is a 33 gauge cannula tip. In some embodiments, the second end includes a one-way check valve integrated into the hub to prevent backflow.

[0024] In certain embodiments, the valve is further coupled to a tubing system. In some embodiments, the cannula tip has an outer diameter ranging from about 0.30 mm to about 0.18 mm. In some embodiments, the cannula tip is fabricated from a flexible polyimide material or metal. In certain embodiments, the cannula tip is 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm in length.

[0025] In some embodiments, the cannula tip is flexible. In certain embodiments, the cannula tip is rigid. In some embodiments, the cannula is attached to an injection device. In certain embodiments, the injection device is a syringe. In certain embodiments, the syringe includes a handle for adjusting the fluid flow path. In certain embodiments, the syringe is a microinjection syringe. In some embodiments, the syringe contains a fluid volume of about 1 mL. In some embodiments, the tubular hub contains a fluid volume of less than 200 μL. In certain embodiments, the tubular hub contains a fluid volume of less than 100 μL. In some embodiments, the tubular hub comprises a fluid volume of about 50 μL (e.g., about 25-50 μL, 50-75 μL, 75-100 μL, 100-150 μL, or 150-200 μL, e.g., about 25, 50, 75, 100, 125, 150, 175, or 200 μL). In some embodiments, the cannula device is further defined as a subretinal delivery device.

[0026] Further embodiments provide for the use of the device in delivering cells to a tissue of a subject. Also provided herein is the use of the device in treating an ocular condition in a subject, comprising administering an effective amount of cells to the eye of the subject. In some aspects, cells are loaded into a cannula and injected into the eye of the subject through the tip of the same cannula. In certain aspects, the cannula tip is not changed between loading into the cannula and injecting into the eye of the subject. In some aspects, cells are delivered from the cell delivery device at a controlled pressure. In certain aspects, cells are not redistributed or resuspended before loading into the cell delivery device. In some aspects, cells are redistributed or resuspended before loading into the cell delivery device. In some aspects, cells are redistributed or resuspended by vortexing or manual stirring. In certain aspects, cells are injected subretinal. In some aspects, a pre-bleb is created through a different cannula before injecting cells through the funnel-shaped hub. In some aspects, cells are further defined as cell aggregates. In certain aspects, the cells are further defined as single cells. In some aspects, the cells are in a formulation buffer. In certain aspects, the formulation buffer is a balanced salt solution. In certain aspects, the balanced salt solution further comprises benzonase and / or human serum albumin.

[0027] In certain embodiments, the cell aggregates are not in suspension when injected into the subject's tissue, hi some embodiments, the cells reside within the cannula for at least 5 minutes between loading and injection to allow for aggregate settling within the cannula hub. In some embodiments, the cells reside within the cannula for at least 1 minute (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, e.g., 1-3, 2-4, 3-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 9-12, 10-13, 11-14, 12-15, 13-16, 15-17, 16-18, 17-19, 18-20, 1-5, 5-10, 10-15, or 15-20 minutes) between loading and injection to allow for settling of the cells, e.g., single cells, within the cannula hub. In certain embodiments, the cannula is oriented downward to allow for settling of the cells, e.g., single cells, within the cannula hub. In certain embodiments, the cell aggregates are photoreceptor progenitor cell aggregates. In certain embodiments, the cells are retinal progenitor cells (RPE) and / or photoreceptor progenitor cells. In some embodiments, the cell aggregates are RPE and / or photoreceptor progenitor cells. In some embodiments, the cells are injected in a volume of less than 200 μL. In some embodiments, the cells are injected in a volume of less than 100 μL. In certain embodiments, the cells are injected in a volume of about 50 μL (e.g., about 25-50 μL, 50-75 μL, 75-100 μL, 100-150 μL, or 150-200 μL, e.g., about 25, 50, 75, 100, 125, 150, 175, or 200 μL). In some embodiments, at least 100,000, 250,000, 500,000, or 1 million cells are injected. In certain embodiments, at least 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, or 15 million cells are injected. In certain embodiments, at least 25% of the cells loaded into the cell delivery device are injected into the subject's eye. In some embodiments, the ocular condition is damage caused by inherited retinal diseases, age-related macular degeneration (AMD), hereditary macular degeneration, Stargardt's macular dystrophy, Best's disease, choroideremia, diabetic retinopathy, retinal vascular disease, retinopathy of prematurity (ROP), or an ocular viral infection.

[0028] Further provided herein are kits comprising the devices and cells of this embodiment and aspects thereof.

[0029] Also provided herein is a method of cell transplantation using an injection attachment having a hub containing a plurality of cells and a cannula extending from a distal end of the hub, the method including orienting the injection attachment so that the cannula faces downward until the plurality of cells settle together adjacent the distal end, inserting the cannula into tissue of a subject, and using the injection attachment to inject the plurality of cells into the tissue of the subject.

[0030] In certain embodiments, the hub includes a funnel-shaped inner surface extending between the distal end and the proximal end. In certain embodiments, the inner surface tapers inwardly from the proximal end to the distal end such that the diameter of the inner surface is smaller at the distal end than at the proximal end. In certain embodiments, the inner surface of the hub does not overlap the cannula.

[0031] In certain embodiments, at least 90% of the cells settle together near the distal end. In certain embodiments, the injection attachment is oriented so that the cannula faces downward for at least 2 minutes. In certain embodiments, the injection attachment is held at an angle of more than 45 degrees while injecting the plurality of cells into the tissue of the subject.

[0032] The method may further include providing a plurality of cells to the injection attachment so that the hub is pre-loaded. In certain aspects, the plurality of cells are drawn into the hub through the distal end of a cannula prior to injection and injected through the cannula. In certain aspects, the cannula is not altered between providing the plurality of cells into the hub and injecting the cells into the tissue of the subject.

[0033] In certain embodiments, the cannula includes a cannula shaft and a cannula tip extending from a distal end of the cannula shaft. In certain embodiments, the cannula tip is a 33 gauge cannula tip. In certain embodiments, the cannula tip is blunt. In certain embodiments, the tissue is retinal tissue. In certain embodiments, the cells are stem cells.

[0034] Also provided herein is a method of cell transplantation in an eye using an injection attachment having a hub containing a plurality of cells and a cannula extending from a distal end of the hub, the method comprising: orienting the injection attachment so that the cannula faces downward until the plurality of cells settle together adjacent the distal end; inserting the cannula into retinal tissue of a subject; and injecting the plurality of cells into retinal tissue of the subject using the injection attachment. In certain embodiments, the injection attachment is oriented so that the cannula faces downward for at least two minutes. In certain embodiments, the hub includes an engagement end and a delivery end and an inner surface extending between the engagement end and the delivery end. In certain embodiments, the inner surface tapers inward between the engagement end and the delivery end. In certain aspects, the plurality of cells are provided in the hub and injected through the cannula.

[0035] Also provided herein is an injection attachment for cell transplantation in an eye, the injection attachment including: a hub having a proximal end, a distal end, and an inner surface extending between the proximal and distal ends and defining an interior volume; and a cannula coupled to the distal end of the hub and in fluid communication with the interior volume. In certain embodiments, the interior volume contains a pre-filled amount of stem cells. In certain embodiments, the interior volume is generally funnel-shaped. In certain embodiments, when the distal end of the hub faces downward, at least 90% of the cells settle proximal to the distal end of the hub. In certain embodiments, the hub includes a body defining the proximal end of the hub and a bushing coupled to the inner surface of the body and defining the distal end of the hub. In certain embodiments, the inner surface tapers inward from the proximal end to the distal end such that the diameter of the inner surface is smaller at the distal end than at the proximal end. In certain embodiments, the interior volume does not retain stem cells after injection.

[0036] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0037] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 is a side view of the injection attachment 100.

[0039] [Figure 2] FIG. 1 is a partial cross-sectional view of an injection attachment 100.

[0040] [Figure 3] FIG. 1 is a flow diagram of rodent and clinical / NHP formulations.

[0041] [Figure 4] A dosing mechanism 200 (e.g., a CONSTELLATION® Vision System) is attached to the tubing of a viscous fluid control pack 210, a MICRODOSE™ injection kit 220, and an injection attachment 100. Optionally, a manual syringe (i.e., a hand-operated plunger) and various cannula tips and cannulas may be used.

[0042] [Figures 5A-5C] For the injection attachment 100, the gauge does not affect the integrity of the cell aggregates. (FIG. 5A) Two users loaded iPRP cell aggregates and discharged them through either a 31 or 33G POLYTIP® cannula to determine if there was any detrimental effect on the aggregate product when passed through a smaller cannula. Neither the percentage of volume within the gate nor the average diameter was clearly affected by the cannula gauge. The percentage of biomass volume above 17.03 diameter was calculated by taking the biomass above 17.03 diameter and dividing by the total biomass (left / primary y-axis). (FIG. 5B) Comparison of the median diameter of iPRP aggregates after either no injection, injection through the injection attachment 100 using a dosing mechanism 200 (e.g., Constellation) to increase and hold the pressure to 10 PSI, then increase to 16 PSI and discharge, or injection at 16 PSI. (FIG. 5C) Comparison of percent aggregate biomass of iPRP aggregates after either no injection, injection through a custom cannula using a dosing mechanism 200 (e.g., Constellation) to increase and hold the pressure to 10 PSI, then increase to 16 PSI and release, or injection at 16 PSI.

[0043] [Figure 6]To ensure consistent dose delivery, iPRP aggregates can be maintained in suspension. Injections 1-34 show the expected cell recovery following a standard FCDI bench test dose injection protocol. The low recovered dose in the surgeon's orientation (injection number 35) was likely due to dose settling within the syringe due to the time and angle at which the syringe was held. Trial number 36 (5-minute rolling dose) shows that the low recovery from the surgeon's orientation was not simply due to the time the dose was held within the syringe. The expected percentage of cells is shown. For example, if 1M cells are recovered, but the 1M dose is the intended target, that is 100% of the expected, whereas if a 2M dose is the intended target and only 1M cells are recovered, the recovery is 50% of the expected.

[0044] [Figure 7] Flicking a syringe loaded with photoreceptor cell aggregates redistributes the settled aggregates, allowing the appropriate dose to be delivered through a standard cannula. Because cell aggregates can be dislodged from the formulation through a standard cannula, we sought to identify a method to maintain appropriate aggregate distribution within the syringe. Figure 7 shows the results of flicking, vortexing, or no mixing with a standard cannula. After dose generation, a standard syringe was prepared and injected with 2 million cells per 50 μL (NHP dose). The syringe was then held in a horizontal position for 5 minutes, after which the dose was mixed or not mixed according to the test conditions. While a clear lack of recovered cells from the no-mixing condition can be seen, the vortexing and flicking condition resulted in recovery of approximately 2 million target cells. Therefore, without effective mixing with a standard cannula, a sufficient dose is not achieved, leaving surgical injection variability.

[0045] [Figures 8A-8F]Simulated injection with the injection attachment 100 and aggregate sedimentation delivers a more consistent dose than the suspension method. (FIG. 8A) Sedimentation of iPRP aggregates within a prefabricated cannula (MedOne #3262) prevented proper delivery of the dose. (FIG. 8B) Technical drawing of a prefabricated 33G POLYTIP® cannula (MedOne #3262). (FIG. 8C) Image of an aggregate (white ring) trapped within the hub of a prefabricated cannula after fully depressing the plunger. Arrows in B and C indicate the cannula hub 301 where the aggregate is trapped. (FIG. 8D) Technical drawing of the funnel-shaped hub 110 of the injection attachment 100. (FIGS. 8E and 8F) Two experiments comparing the injection attachment 100 with a prefabricated cannula demonstrate that the sedimentation method delivers a more consistent dose than the suspension method.

[0046] [Figure 9] Passing iPRP aggregates through a 33G cannula twice does not significantly affect aggregate size compared to a single pass. Two users loaded the dose into a syringe through an 18G needle (single pass) or a 33G cannula (double pass) and then expelled photoreceptor cell aggregates through a standard 33G POLYTIP® cannula. These aggregates were then analyzed by Multisizer to determine any impact on aggregate integrity. Neither the percentage of volume within the gate nor the average diameter was significantly affected by the number of times the aggregates passed through the cannula.

[0047] [Figure 10] Schematic of dose preparation for concentration studies. iPRP aggregate cells were thawed, washed, and then resuspended at a concentration higher than the target concentration. A sample from this bulk was taken for cell counting. Once the bulk concentration was determined, it was dispensed into separate tubes and sufficient vehicle was added to dilute the master dose to the final concentration. These master doses were then dispensed into aliquots intended for injection.

[0048] [Figure 11]Master Dose Variability from Concentration Studies. The percent expected recovery from iPRP aggregates of 28 different master doses across 10 experiments averaged 93% at injection attachment 100, with a coefficient of variation of 11.4%.

[0049] [Figure 12] Schematic diagram of final dose preparation. iPRP aggregate cells were thawed, washed, and then resuspended at a concentration higher than the target concentration. A sample was taken for cell count, and the bulk volume (minus 20 μL) was transferred to a separate tube and sufficient vehicle was added to dilute the master dose to the final concentration. This master dose was then dispensed into aliquots intended for injection.

[0050] [Figure 13] Master Dose Variability Generated from Clinical Workflow. Master doses of iPRP aggregates produced by the clinical workflow (1 bulk to 1 master dose) averaged 93.5% of the expected cell concentration, with a coefficient of variation of 5.0%.

[0051] [Figure 14] The dose-to-dose coefficient of variation for doses administered ranged from 7.5% to 15.5%. In eight experiments with the injection attachment 100, where master doses were generated from a single bulk preparation of iPRP aggregates, 5 and 12 aliquots ranging in volume from 60 μL to 210 μL were sampled and dissociated for counting, and the % expected recovery was calculated to determine dose-to-dose variability. All doses were within ±55% of the expected cell concentration.

[0052] [Figures 15A-15C]Dose recovery for various cell concentrations. After observing lower than expected recovery of injected cells, calculated doses were examined across various concentrations using the injection attachment 100. (Figure 15A) After preparing 82 calculated doses across 11 experiments and 6 lots, it was determined that 1.7 million cell doses were required to inject 1 million cells (clinical dose) and 2.8 million cell doses were required to inject 2 million cells (NHP dose). (Figure 15B) For the 1.7 million cell dose, across 6 experiments, 3 lots, and 25 injections, the average delivered injection contained 1.04 million cells, with a coefficient of variation of 19%. 84% (21 / 25) of the doses fell within ±30% of the 1 million cell target. (Figure 15C) For the 2.8 million cell dose, across 3 experiments, 3 lots, and 15 injections, the average delivered injection contained 2.1 million cells, with a coefficient of variation of 16%. Ninety-three percent (14 / 15) of the doses fell within ±30% of the 2 million cell target. For the 3 and 4 million cell doses, a linear fit line was extrapolated across all data points generated for 391 individual injections across two iPSC lines, eight iPRP lots (16 sublots) for all four doses (best fit equation y=0.0348x-0.1239). Using this equation, it was calculated that doses of 4.5 million and 5.95 million cells would need to be loaded to achieve the target doses of 3 million and 4 million cells injected, respectively.

[0053] [Figures 16A-16C]Immunofluorescence images demonstrating the viability of transplanted cells and photoreceptor identity in injections into the eye above the fovea. (Figures 16A-16C) Representative photomicrographs of the transplanted cell region (multiple regions are imaged). Stem121 labels the cytoplasm of all transplanted human cells (note that the host RPE is also highly autofluorescent). AIPL1 labels rod and cone photoreceptors. There is some cross-reactivity of AIPL1 with host NHP photoreceptors. However, the labeling of transplanted human photoreceptors is much brighter. In normal retinas with an intact ONL and outer limiting membrane, as expected, iPRP cell aggregates reside primarily within the subretinal space and do not integrate into the host ONL. A'-C') Corresponding brightfield (DIC) images. Transplanted cells often made contact with pigmented RPE (brownish cells surrounding the AIPL1 / Stem121 staining). It was unclear whether this was an artifact of thick cryosectioning or a biological response to the xenograft. Note that the separation of the neural retina is an artifact of tissue processing and sectioning. T = transplanted cell area. ONL = outer nuclear layer. INL = inner nuclear layer.

[0054] [Figure 17A] 2 is a side cross-sectional view of the injection attachment 100 of FIG. 1 coupled to a syringe, containing a sedimentary volume of cells within the distal end of the funnel-shaped hub 110. FIG.

[0055] [Figure 17B] 1 is a side cross-sectional view of the injection attachment 100 of FIG. 1 connected to a syringe containing a sedimentary volume of cells within the distal end of the funnel-shaped hub and inserted into the eye of a subject 110. FIG. DETAILED DESCRIPTION OF THE INVENTION

[0056] There is an unmet need for improved methods and devices for delivering cell compositions that reliably deliver precise, high-density doses to target tissues without cell damage or loss. The integrity of cell compositions can be adversely affected by how the cell composition is handled, such as through multiple handling steps involving needle exchange. Cell compositions can include single-cell suspensions or cell aggregates, and can be single or multicellular. Cells in the composition can settle out of the formulation buffer, which can affect dose formulation and delivery. In particular, accurate dose delivery cannot be reliably achieved using standard cannulas because cells in the composition can become trapped within the cannula hub due to settling and improper resuspension techniques. Therefore, we hypothesized that custom cannulas with funnel-shaped hubs would improve the consistency of injected doses because there is essentially no space for single cells or aggregates of cell compositions to become trapped.

[0057] In some embodiments, the cell aggregates can comprise a median aggregate diameter of about 40-60 μm, e.g., 40-45, 45-55, 50-55, or 50-60 μm. Aggregate integrity can be determined by measuring the aggregate diameter and the percentage of aggregates >17.03 μm. In some embodiments, the aggregate size can be 10-200 μm, e.g., 15-170 or 17-168 μm.

[0058] With the knowledge that cell compositions, such as cell aggregates, can fall out of the formulation buffer when loaded into a syringe, potentially resulting in an inaccurate injected dose, we sought to identify a method to maintain proper aggregate distribution within the syringe. To prevent aggregate settling, flicking and vortexing were tested and shown to be effective for mixing with a standard cannula (Figure 7). This "resuspension" method involves drawing an excess volume into a syringe attached to a cannula, flicking the unnecessary volume to remove air bubbles, and then injecting the remaining volume. The problem with this method is that cells, such as aggregates, may not be evenly distributed throughout the volume and may settle. Therefore, resuspension methods can result in an inhomogeneous cell suspension, leading to overdosing or underdosing. Furthermore, even with flicking or vortexing, loading and injection must be performed quickly, and even then, variability in the cell composition delivered during surgical injection persists.

[0059] Therefore, we investigated whether intentionally settling aggregates would allow for more consistent dose injection. However, studies with standard cannulae showed that syringes that allowed cells to settle injected less than 25% of the expected dose. It was found that the standard syringe (FIG. 8B) delivered a lower dose because it contained a cannula hub 301 that could trap cells and prevent them from being injected.

[0060] Because settling of aggregates toward the cannula tip initially did not work due to the geometry of the cannula hub, the injection attachment 100, which uses a new, more funnel-like hub and cannula extending distally from the hub—referred to as a cannula or custom cannula design in the applications from which this disclosure claims priority—achieves much more consistent doses. The cannula may include a continuous funnel-like flow pattern that reduces resistance and turbulence as the liquid passes through the cannula. This newly designed cannula allows for smaller doses (e.g., 50 μl) to be loaded through the cannula and allowed to settle toward its tip for 5 minutes before injection, eliminating the need to keep the aggregates suspended prior to injection. The settling method used by the injection attachment 100 has also been shown to reduce the variability of administered doses. Specifically, the shape and settling method of the injection attachment 100 have been shown to enable improved precision. With improved accuracy, dead volume can be overcome by using a correction factor to accurately administer the dose (i.e., the correct number of cells). The percent improvement is shown in Figure 6, where the dose is within 40-90% of the expected range compared to the surgeon-directed dose, which has a recovery rate of less than 20%. Therefore, the present cannula and method may enable delivery of the complete dose of the cell composition.

[0061] In certain embodiments, the "sedimentation" method precisely delivers a targeted dose by preventing cells from becoming trapped in the cannula. Specifically, in this sedimentation method, the dose is flicked to resuspend cells, such as aggregates, and then drawn into a pre-loaded syringe attached to a cannula, which then enters the device through the same cannula, which is then used to withdraw and implant the cells into an injection site, such as the subretinal space. In certain embodiments, the syringe attached to the cannula with the loaded dose is held vertically for approximately 2 to 15 minutes to allow cells, such as cell aggregates, to settle before injecting the dose.

[0062] Thus, in certain embodiments, provided herein are devices and methods of using the devices that enable reliable delivery of cell compositions by providing an injection attachment 100 having a funnel-shaped hub 110. In certain aspects, the present disclosure provides cell delivery devices that enable reliable dose delivery of cell compositions (e.g., compositions of single cells or aggregated cells). In certain aspects, the injection attachment 100 enables improved accuracy of dose delivery, with accuracy further improved by implementing a dose correction factor. Thus, in certain embodiments, provided herein are methods of delivering cell therapies, such as aggregated iPSC-derived PRP cell therapies.

[0063] In addition to improving dose consistency, in the present method, the injection attachment 100 described herein allows for a reduction in dead volume of cell solution compared to previous methods. For example, the injection attachment 100 described herein may allow for the aspiration of 50 μl of cell solution into a cannula for injection to deliver a 50 μl dose, whereas previous cannulas require the aspiration of 200 μL of cell solution for injection to deliver a 50 μl dose.

[0064] Furthermore, in certain embodiments, the present injection attachment 100 includes a smaller diameter (i.e., larger gauge) compared to standard cannulas. Specifically, previous devices include cannulas with larger diameters (e.g., 31G). The larger the cannulas used, the lower the shear forces during loading and injection. However, contrary to previous expectations that smaller diameters would cause cell damage or loss, the present study demonstrated that smaller diameter cannulas (e.g., 33G) do not adversely affect cell compositions, such as cell aggregate compositions. Specifically, the initial shear forces exerted on the cell compositions were shown to have no effect on the cell compositions, particularly due to the funnel-shaped hub of the present injection attachment 100. Additionally, smaller cannula diameters offer the advantage of minimizing the amount of trauma at the injection site and providing better surgical outcomes with less reflux of the cell compositions.

[0065] In certain embodiments, the hub is essentially free of catch points that can increase shear or turbulence as cells move through the hub. The hub is substantially smooth as cells pass through the hub and into the cannula shaft. Reducing turbulent shear stress helps improve cell viability, while catch points cause cell attrition and damage. Therefore, the design of the injection attachment 100 allows for reduced cell loss.

[0066] Thus, in some embodiments, the device includes an injection attachment 100 coupled to a dosing mechanism 200 or manual syringe, such as those from CONSTELLATION®, STELLARIS®, and EVA® Vision Systems. The injection attachment 100 includes a funnel-shaped hub 110, which prevents any settling or trapping of cells in the hub, allowing for higher doses of cells to be delivered to the subject. The cannula tip 121 can be a 33-gauge or 31-gauge cannula tip. The method allows for the use of smaller dose volumes, such as less than 200 μL, less than 175 μL, less than 150 μL, less than 100 μL, less than 75 μL, less than 50 μL, less than 25 μL, or less than 10 μL. For example, the hub can include an internal volume 117 of less than 200 μL, less than 175 μL, less than 150 μL, less than 100 μL, less than 75 μL, less than 50 μL, less than 25 μL, or even less than 10 μL. The hub can include an internal volume of about 10-25 μL, 25-50 μL, 50-75 μL, 75-100 μL, 100-150 μL, 150-175 μL, 175-200 μL, or 200-250 μL. In some embodiments, the hub 110 can include an internal volume 117 of greater than 5 μL, 10 μL, 25 μL, 50 μL, 75 μL, 100 μL, 150 μL, 175 μL, or 200 μL.

[0067] The cell composition may contain various excipients (e.g., buffers, salts, polymers, proteins, and preservatives) to, for example, stabilize the cells or provide physiological osmolality. The cells may be suspended in a formulation buffer. The buffer may be a phosphate buffer, a citrate buffer, an acetate buffer, or another organic acid buffer. The buffer may contain DMSO, benzonase, or albumin. In certain embodiments, the buffer is a balanced salt solution (BSS), such as a BSS containing DMSO or albumin, or a specific human serum albumin (HSA), such as a 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% HSA buffer. The cells may be resuspended before loading, for example, by vortexing or manually flicking the vial containing the cell composition. In some embodiments, as shown in FIG. 17A, cells, such as cell aggregates, are allowed to settle in the cannula for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, particularly 5 minutes, after loading and before injection. In certain embodiments, the cells in the composition are allowed to settle within the cannula after loading and before injection, e.g., for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In certain embodiments, the cells in the composition are allowed to settle within the cannula after loading and before injection, e.g., for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In certain embodiments, the cells in the composition are allowed to settle within the cannula after loading and before injection, e.g., for about 1-3, 2-5, 3-6, 4-7, 5-8, 6-9, or 7-10 minutes. In certain embodiments, the cells in the composition are allowed to settle within the cannula after loading and before injection, e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In certain embodiments, the cells in the composition are allowed to settle within the cannula after loading and before injection, eg, for at least 1-3, 2-5, 3-6, 4-7, 5-8, 6-9, or 7-10 minutes.

[0068] Previously, a single-pass method was used, which involved loading the cell composition through a first larger needle, such as an 18G needle, and then exchanging the larger diameter needle for a smaller diameter needle, such as a 31G cannula, prior to injection. Generally, larger diameter needles are used for loading to reduce shear forces during loading, and smaller diameter needles are used for injection to minimize the amount of damage or pain at the injection site. In the single-pass method, a larger dose (e.g., 200 μL) is loaded into the syringe to facilitate removal of air within the syringe and cannula. Once the air is removed, the next smaller dose (e.g., 50 μL) is dispensed until a smaller dose remains. The single-pass method requires a needle change, which can result in loss of integrity and damage to the cell composition and variability in the administered dose.

[0069] However, in certain embodiments, the present method relates to a dual-pass method in which the needle is not changed between loading and injection. This study showed that aggregates can be passed through the cannula twice without resulting damage (Figure 9). This dual-pass method also allows for overhaul of syringe handling before dose delivery. In this dual-pass method, cells are loaded and released through the same cannula, which reduces handling steps and reduces the chance of contamination and needle sticks. Notably, with the dual-mode method, the same volume of dose (e.g., 50 μL) can be loaded and injected through a single needle compared to the larger initial volume used in the single-pass method.

[0070] Therefore, in certain embodiments, a delivery device and method for delivering cell therapy, such as PRP cell aggregates, are provided herein. The method allows for consistent, high-dose cell delivery without significant cell loss and at lower dosages. In certain aspects, the cell therapy can be delivered subretinal to the subject's eye using the device, which includes a cannula with a funnel-shaped hub. In some aspects, a bleb is inserted into the retina before administering the cells.

[0071] II. Definition The term "purified" does not require absolute purity. Rather, it is intended as a relative term. Thus, a purified cell population is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, or most preferably essentially free of other cell types.

[0072] As used herein, "a" or "an" may mean one or more. When used in conjunction with the word "comprise," the words "a" or "an" used in the claims may mean one or more.

[0073] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, although the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" may mean at least a second or more.

[0074] The term "essentially" should be understood to include only those methods or compositions that do not materially affect the specified steps or materials and the basic and novel characteristics of those methods and compositions.

[0075] As used herein, a composition or medium that is "substantially free" of a particular substance or material contains ≦30%, ≦20%, ≦15%, more preferably ≦10%, even more preferably ≦5%, or most preferably ≦1% of the substance or material.

[0076] As used herein, the terms "substantially" or "approximately" may be used to modify any quantitative comparison, value, measurement, or other expression that can vary acceptably without resulting in a change in the basic function to which it pertains.

[0077] The term "about" generally means within the standard deviation of the stated value as determined using standard analytical techniques to measure the stated value. The term may also be used to refer to ±5% of the stated value.

[0078] As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the particular component is intentionally incorporated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the particular component can be detected by standard analytical methods.

[0079] The term "stem cell" as used herein refers to a cell that, under appropriate conditions, can differentiate into a diverse range of specialized cell types, while under other appropriate conditions, it can self-renew and maintain an essentially undifferentiated, pluripotent state. The term "stem cell" also encompasses pluripotent, multipotent, progenitor, and progenitor cells. Exemplary human stem cells can be derived from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be generated from somatic cells by reprogramming them to a pluripotent state through the expression of specific transcription factors associated with pluripotency. These cells are referred to as "induced pluripotent stem cells" or "iPSCs."

[0080] The term "pluripotency" refers to the property of a cell to differentiate into all other cell types in an organism, except for extraembryonic or placental cells. Pluripotent stem cells can differentiate into cell types of all three germ layers (e.g., ectodermal, mesodermal, and endodermal cell types), even after long-term culture. Pluripotent stem cells are embryonic stem cells derived from the inner cell mass of a blastocyst. In another embodiment, the pluripotent stem cells are induced pluripotent stem cells derived by reprogramming somatic cells.

[0081] The term "differentiation" refers to the process by which unspecialized cells become more specialized types with altered structural and / or functional properties. Mature cells typically have altered cellular structures and tissue-specific proteins.

[0082] As used herein, "undifferentiated" refers to cells that display characteristic markers and morphological features of undifferentiated cells that clearly distinguish them from terminally differentiated cells of embryonic or adult origin.

[0083] Embryoid bodies (EBs) are aggregates of pluripotent stem cells that can undergo differentiation into endoderm, mesoderm, and ectoderm cells. EBs are formed in suspension by aggregating pluripotent stem cells under non-adherent culture conditions, resulting in the formation of spheroid structures.

[0084] An "isolated" cell is one that has been substantially separated or purified from other cells in an organism or culture. An isolated cell can be, for example, at least 99%, at least 98% pure, at least 95% pure, or at least 90% pure.

[0085] "Embryo" refers to a mass of cells resulting from one or more divisions of a zygote or activated oocyte having an artificially reprogrammed nucleus.

[0086] "Embryonic stem (ES) cells" are obtained from an early stage embryo, such as the inner cell mass of the blastocyst stage, or are generated by artificial means (e.g., nuclear transfer), and are undifferentiated pluripotent cells that can give rise to any differentiated cell type, including germ cells (e.g., sperm and eggs), in the embryo or adult.

[0087] "Induced pluripotent stem cells (iPSCs)" are cells generated by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram the somatic cell into a pluripotent stem cell.

[0088] "Allele" refers to one of two or more forms of a gene. Diploid organisms such as humans contain two copies of each chromosome, and therefore have one allele of each.

[0089] The term "homozygous" is defined as containing two identical alleles at a particular locus. The term "heterozygous" refers to containing two different alleles at a particular locus.

[0090] A "haplotype" refers to a combination of alleles at multiple loci along a single chromosome. A haplotype can be based on single nucleotide polymorphisms (SNPs) on a single chromosome and / or a set of alleles in the major histocompatibility complex.

[0091] As used herein, the term "haplotype-matched" is defined as cells (e.g., iPS cells) and the subject being treated sharing one or more major histocompatibility locus haplotypes. A subject's haplotype can be readily determined using assays well known in the art. Haplotype-matched iPS cells can be autologous or allogeneic. Autologous cells expanded in tissue culture and essentially differentiated into PRP cells are haplotype-matched to the subject.

[0092] "Substantially the same HLA type" indicates that the donor's human leukocyte antigen (HLA) type matches that of the patient to the extent that transplanted cells obtained by inducing differentiation of iPSCs derived from the donor's somatic cells can engraft when transplanted into the patient.

[0093] "Superdonor," as used herein, refers to an individual who is homozygous for particular MHC class I and II genes. These homozygous individuals can serve as superdonors, and their cells (including tissues and other materials containing the cells) can be transplanted into individuals who are either homozygous or heterozygous for that haplotype. Superdonors can be homozygous for each of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ loci / locus alleles.

[0094] "Feeder-free" or "feeder-independent" is used herein to refer to cultures supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF) as an alternative to a feeder cell layer. Thus, "feeder-free" or feeder-independent culture systems and media can be used to culture and maintain pluripotent cells in an undifferentiated and proliferative state. Optionally, feeder-free cultures utilize an animal-based matrix (e.g., MATRIGEL™) or are grown on substrates such as fibronectin, collagen, or vitronectin. These approaches allow human stem cells to remain essentially undifferentiated without the need for a "feeder layer" of mouse fibroblasts.

[0095] A "feeder layer" is defined herein as a coating layer of cells, such as on the bottom of a culture dish. Feeder cells may release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, can attach.

[0096] The terms "defined" or "fully defined," when used with respect to a medium, extracellular matrix, or culture condition, refer to a medium, extracellular matrix, or culture condition in which the chemical composition and amount of nearly all components are known. For example, a defined medium does not contain undefined factors such as fetal bovine serum, bovine serum albumin, or human serum albumin. Generally, a defined medium comprises a basal medium (e.g., Dulbecco's Modified Eagle's Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640 containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and an energy source) supplemented with recombinant albumin, chemically defined lipids, and recombinant insulin. An example of a fully defined medium is Essential 8™ medium.

[0097] For media, extracellular matrices, or culture systems used with human cells, the term "xeno-free (XF)" refers to a condition in which the materials used are not of non-human animal origin.

[0098] "Preconfluent" refers to a cell culture in which the percentage of the culture surface covered by cells is approximately 60-80%. Typically, preconfluent refers to a culture in which approximately 70% of the culture surface is covered by cells.

[0099] The term "neural retinal progenitor" or "NRP" refers to cells that have restricted potential to differentiate into neural retinal cell types.

[0100] The term "photoreceptor" or "PR" cell refers to a cell that is within the photoreceptor lineage (i.e., maturation) pathway both before and after upregulation of expression of rhodopsin (rods) or any of the three cone opsins (cones), which encompasses both early and late markers of photoreceptor cells (rods, cones, or both).

[0101] The term "photoreceptor progenitor cells" or "PRPs" refers to cells differentiated from embryonic stem cells or induced pluripotent stem cells that can differentiate into photoreceptor cells that express the cell marker rhodopsin or any of the three cone opsins. The photoreceptors can be rod and / or cone photoreceptors.

[0102] The term "retinal degeneration-related disease" is intended to refer to any disease resulting from congenital or postnatal retinal degeneration or abnormality. Examples of retinal degeneration-related diseases include retinal dysplasia, retinal degeneration, age-related macular degeneration, Stargardt's disease, Best's disease, choroideremia, hereditary macular degeneration, myopic degeneration, RPE rupture, macular hole, diabetic retinopathy, retinitis pigmentosa, hereditary retinal diseases or degenerations, hereditary macular degeneration, cone-rod dystrophy, rod-cone dystrophy, congenital retinal dystrophy, Leber's congenital amaurosis, retinal detachment, and retinal trauma.

[0103] As used herein, the term "ocular condition" refers to a disease, condition, illness, or injury of the ocular region. In some embodiments, the ocular condition may include a condition in the posterior segment of the eye. In other embodiments, the ocular condition may include a condition in the anterior segment of the eye. The ocular condition may be associated with retinal epithelial cells and / or photoreceptor cells. The ocular condition may be related to or associated with diabetes (e.g., diabetic macular edema, retinal artery occlusive disease, or diabetic retinopathy), age (e.g., age-related macular degeneration, choroidal neovascularization, subretinal fibrosis, or glaucoma), inflammation (e.g., Behçet's disease, posterior uveitis, serous choroiditis, uveitis syndrome, cytomegalovirus retinitis, or endophthalmitis), genetics (e.g., Coats disease or familial exudative vitreoretinopathy), or cancer (e.g., posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, mixed hamartoma of the retina and retinal pigment epithelium, retinoblastoma, angioproliferative tumor of the fundus, retinal astrocytoma, retinal carcinoma, or intraocular lymphoid tumor). The disease may be a retinopathic condition, such as acute and chronic macular neuroretinopathy, or central serous chorioretinopathy. For example, ocular conditions include, but are not limited to, age-related macular degeneration, choroidal neovascularization, diabetic macular edema, acute and chronic macular neuroretinopathy, central serous chorioretinopathy, macular edema, acute multifocal disc pigment epitheliopathy, Behcet's disease, birdshot retinochoroidopathy, posterior uveitis, posterior scleritis, serous choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusion, central retinal vein occlusion, disseminated intravascular coagulation, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal arterial microaneurysms, Coats disease, parafoveal telangiectasia, and hemiretinal vein occlusion. , papillophlebitis, carotid artery disease (CAD), chilblain bifurcation vasculitis, sickle cell retinopathy, vascularized striatum, familial exudative vitreoretinopathy, Eales' disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor-associated retinal diseases, congenital hypertrophy of the retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, mixed hamartoma of the retina and retinal pigment epithelium, retinoblastoma, angioproliferative tumor of the fundus, retinal astrocytoma, intraocular lymphoma, myopic retinal degeneration, acute retinal pigment epitheliitis, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal cancer, and any combination thereof. Injuries to the eye may include contusion, puncture, scratch, penetrating injury, perforation injury, or injury resulting from an intraocular foreign body.

[0104] As used herein, a "therapeutically effective amount" refers to the amount of a compound that, when administered to a subject for treating a disease or condition, is sufficient to affect such treatment.

[0105] An "inducer" is defined herein as a molecule that regulates gene expression, such as activating a gene in a cell. An inducer can bind to a repressor or an activator. An inducer functions by disabling a repressor.

[0106] As used herein, the term "engrafted" bilayer refers to transplanted cells engrafting into the host retina and forming pre- and post-synaptic mechanisms such that the transplanted and host cells are poised to form synapses.

[0107] As used herein, the term "biodegradable" refers to a material that provides initial structural support for delivered cells, but degrades over time into products that are not toxic to the transplant host and do not contribute to donor site morbidity.

[0108] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate, particularly a human. Non-limiting examples of human patients include adults, juveniles, infants, and fetuses.

[0109] As used herein, "smooth" refers to a cannula hub that has essentially no protrusions projecting inward from the inner surface of the hub.

[0110] III. Cell delivery device In some aspects, the present disclosure provides an apparatus for delivering a composition of cells. The delivery device may include an injection attachment having a cannula and a funnel-shaped hub, such as injection attachment 100 of FIGS. 1-2. The cannula at distal end 112 of hub 110 of FIG. 1 may have an outer diameter of less than 0.3 mm, e.g., in the range of 0.15 mm to 0.3 mm, 0.20 mm to 0.25 mm, 0.25 mm to 0.3 mm, or 0.15 mm to 0.25 mm. Cannula hub 110, such as distal end 112 of FIG. 1, may be a 22-gauge, 23-gauge, 24-gauge, 25-gauge, 26-gauge, 27-gauge, or 28-gauge cannula hub. For example, a microcannula may be used. Injection attachment 100 may have a cannula shaft, such as a 25-gauge cannula (e.g., cannula 120 of FIG. 1). At the distal end of cannula 120, the shaft tapers to a smaller cannula tip 121 attached to cannula 120. Cannula 120 can be provided with a small gauge tip tapered design (e.g., 121 in FIG. 1), such as an outer diameter ranging from about 0.30 mm to 0.18 mm or less. Cannula tip 121 can have an outer diameter of approximately 0.1 mm, 0.12 mm, 0.14 mm, 0.16 mm, 0.18 mm, 0.2 mm, 0.22 mm, 0.24 mm, 0.26 mm, 0.28 mm, or 0.3 mm. The cannula tip 121 can be a 30 gauge (e.g., 0.30 mm diameter), 31 gauge, 32 gauge, 33 gauge, 34 gauge (e.g., 0.18 mm diameter), 35 gauge, 36 gauge, 37 gauge, 38 gauge (e.g., 0.12 mm diameter), 39 gauge, or 40 gauge (e.g., 0.10 mm diameter) cannula tip, particularly a 30-34 gauge cannula tip having a diameter of about 0.30 mm to about 0.18 mm. For example, the cannula 120 can be a 25 gauge cannula 120 having a 33 gauge cannula tip 121. Additionally, the cannula tip 121 can be fabricated from flexible polyimide, metal, or other similar materials. The tip can include a length of 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm and can be rigid or flexible and extendable.

[0111] The injection attachment 100 can be attached to a syringe 220, such as a syringe that allows for controlled microinjection, such as a MICRODOSE™ injector. The injection attachment 100 can include a connection mechanism 113, such as a luer lock hub, or a connection to the syringe 220, extension tubing 230, or infusion line 240. The tubing can be silicone tubing, such as silicone tubing. The device can further include a viscous fluid control pack 210. In other embodiments, the tubing 230 can be packed with the syringe 220, such as a MICRODOSE™ injector, without the viscous fluid control pack 210.

[0112] Embodiments of the present disclosure include methods of use that include a cannula. Referring now to Figures 1-2, there is shown an injection attachment 100 that includes a funnel-shaped hub 110, a cannula 120, and a cannula tip 121. Figure 1 shows a side view of injection attachment 100, while Figure 2 shows a partial cross-sectional view taken along line 2-2 of Figure 1.

[0113] In an exemplary embodiment, the coupling mechanism 113, such as a luer lock hub, may be coupled to a dispensing mechanism 200 (not shown), including, for example, a CONSTELLATION® Vision System or a manual syringe. It is understood that the listed dispensing mechanisms are merely exemplary of certain embodiments, and that other embodiments according to the present disclosure may be coupled to any suitable dispensing mechanism.

[0114] 2 , in the illustrated embodiment, funnel-shaped hub 110 includes a proximal end 111, referred to herein as the first end in the applications to which this disclosure claims priority, a distal end 112, referred to herein as the second end in the applications to which this disclosure claims priority, and an interior volume 117 defined by an interior surface 115. The term “funnel-shaped” as used herein may be broadly defined as the interior volume 117 configured to direct particles (e.g., cells suspended in a fluid) through hub 110 and into cannula 120 while minimizing or eliminating the presence of retained particles. In certain embodiments, proximal end 111 may include threads (e.g., coupling mechanism 113) or other suitable coupling mechanism (e.g., including a “Luer Lock” type design) configured to engage a dispensing mechanism 200. Coupling mechanism 113 may include an interior surface configured to couple to the exterior surface of dispensing mechanism 200. The coupling mechanism 113 may include an angled protrusion that is received by a corresponding protrusion on the dispensing mechanism 200, for example, to couple via a threaded engagement. The coupling mechanism 113 and the dispensing mechanism 200 may be coupled via a snap fit, adhesive, epoxy, welding, or other engagement sufficient to prevent unintentional detachment of the coupling mechanism 113 and the dispensing mechanism 200. In use, the dispensing mechanism can transfer contents from the internal volume 117 through the cannula 120 and cannula tip 121 to a subject, as shown in FIG. 17B.

[0115] As shown in FIG. 2, the inner surface 115 can be generally funnel-shaped. The funnel shape helps minimize particle retention after injection. The inner surface 115 tapers continuously from the proximal end 111 (e.g., the engagement end) to the distal end 112 (e.g., the delivery end). The taper can have a constant angle between the proximal end 111 and the second end 122. As shown in FIG. 2, the taper can have a variable angle between the proximal end 111 and the second end 122. In the example shown, the inner diameter D1 (measured across the inner surface 115 within the interior volume 117) decreases as measured from the proximal end 111 to the distal end 112. While the sides shown in FIG. 2 are mirror images of each other, it is contemplated that the inner surface 115 can taper toward one side or can taper inwardly and outwardly between the proximal end 111 and the distal end 112. In some embodiments, one side of the inner surface 115 may be flat. In this manner, the inner surface 115 does not include any protrusions or other features that may restrict the contents of the interior volume 117 from exiting the hub 110 and entering the cannula 120. Therefore, the contents of the interior volume 117 (e.g., a suspension of cells, therapeutic agent, or other suitable contents) can move freely from the proximal end 111 to the distal end 112 of the injection attachment 100, and then further into the cannula 120 and tip 121. The method and injection attachment 100 may result in at least 99% of the cells contained within the injection attachment 100 being injected into tissue. In some embodiments, at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% of the cells contained within the injection attachment 100 are injected into tissue. The continuous taper of inner surface 115 and the lack of protrusions into interior volume 117 also allows the contents of the interior volume to be thoroughly mixed prior to administration to a subject.

[0116] In certain embodiments, cannula 120, referred to herein and referred to as SIFT in the application from which this disclosure claims priority, can be configured such that the diameter of its shaft does not restrict or limit the dose of infusate provided by injection attachment 100. In certain embodiments, cannula 120 can be configured as a 25-gauge cannula or other suitable diameter shaft. In certain embodiments, cannula tip 121 can be configured as a 33-gauge (33G) cannula, 31-gauge (31G) cannula, or other suitable diameter tip. Thus, the combination of hub 110 (without protrusions or restrictions on inner surface 115) and cannula 120 of an appropriate diameter allows injection attachment 100 to thoroughly mix the contents of internal volume 117 and administer the contents within internal volume 117 without restriction. Thus, the contents of internal volume 117 can be administered to a subject in accurate and repeatable doses.

[0117] IV. Cell delivery method In certain aspects, the present disclosure provides methods for cell transplantation, such as cell aggregate or single cell suspension therapy. The methods can be used for a variety of cell transplantation procedures known in the art. The transplantation can include providing a quantity of cells to a cell delivery device (not shown), the device including a proximal end and a distal end opposite the proximal end. The device can include an interior volume configured to receive the quantity of cells.

[0118] The device may include a cannula (e.g., cannula 120) extending from funnel-shaped hub 110. The cannula may include a cannula tip 121. Cannula tip 121 may be a 33 gauge cannula tip. In some embodiments, cannula tip 121 is a 32 gauge cannula tip, a 31 gauge cannula tip, a 30 gauge cannula tip, a 29 gauge cannula tip, a 28 gauge cannula tip, a 27 gauge cannula tip, a 26 gauge cannula tip, a 25 gauge cannula tip, a 24 gauge cannula tip, a 23 gauge cannula tip, or a 22 gauge cannula tip.

[0119] A funnel-shaped hub 110 is coupled to the distal end of the device and is in fluid communication with the interior volume. The funnel-shaped hub 110 may include a proximal end 111 and a distal end 112, with an inner surface 115 extending between the proximal end 111 and the distal end 112. The inner surface 115 may be generally cylindrical between the proximal end 111 and the distal end 112.

[0120] The inner surface 115 may have a diameter at the distal end 112 that is smaller than the diameter of the inner wall of the proximal end 111. The inner surface 115 may be tapered between the proximal end 111 and the distal end 112. A cannula (e.g., cannula 120) may be coupled to the proximal end 111 of the funnel-shaped hub 110. The cannula 120 is coupled to the distal end 112 using an adhesive or a suitable epoxy. In some embodiments, the cannula 120 is integrally coupled to the distal end 112. The cannula 120 does not extend beyond the distal end 112 of the funnel-shaped hub 110. As shown in FIG. 2 , the cannula 120 is coupled to the distal end 112 such that no portion of the cannula 120 enters the interior volume 117.

[0121] The device can be oriented so that the distal end is lower than the proximal end relative to the ground for a period of time. This period of time can be sufficient for the cells to settle to the distal end of the hub 110. Sedimentation can refer to approximately 90% of the cells settling into the hub 110, as shown in FIG. 17A. In some embodiments, sedimentation refers to at least 50% of the cells, at least 60% of the cells, at least 70% of the cells, at least 80% of the cells, at least 90% of the cells, at least 95% of the cells, or at least 99% of the cells. In some embodiments, sedimentation refers to 50%-90% of the cells, 55%-85% of the cells, 60%-80% of the cells, or 65%-75% of the cells.

[0122] This period of time may be sufficient for the cells to settle to the distal end 112 of the funnel-shaped hub 110. As shown in FIGS. 17A-17B, cells (C) may settle at the distal end 112 of the funnel-shaped hub 110. Also shown in FIGS. 17A-17B is the liquid solution (S) in which the cells C are suspended. The funnel-shaped hub 110 may include a transparent material to allow visual inspection of the cells settling therein. This period of time may be between 30 seconds and 10 minutes. This period of time may be between 1 minute and 9 minutes, between 2 minutes and 8 minutes, between 3 minutes and 7 minutes, or between 4 minutes and 6 minutes. This period of time may be at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, or at least 10 minutes. In certain embodiments, centrifugal force may be applied to the hub to shorten the period of time required for the cells to settle proximate the distal end of the hub.

[0123] The method may include inserting a cannula into tissue of a subject. The cannula tip 121 may be blunt and configured to enter the tissue through an incision. In some embodiments, the cannula tip 121 may be sharp and configured to pierce and enter the tissue. The tissue may be retinal tissue.

[0124] As shown in FIG. 17B, the device can be held at an angle (A) while injecting cells into the target tissue. The device can be held at an angle A of approximately 45 degrees relative to the ground during injection. The device can be held at an angle A of approximately 20 degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, 50 degrees, 55 degrees, 60 degrees, 65 degrees, or 70 degrees relative to the ground during injection. The device can be held at an angle A of 20 degrees to 70 degrees relative to the ground during injection. The device can be held at an angle A of 25 degrees to 65 degrees, 30 degrees to 60 degrees, 35 degrees to 55 degrees, 40 degrees to 50 degrees, or 45 degrees to 55 degrees relative to the ground during injection.

[0125] The method may include injecting the quantity of cells into the tissue of the subject using a cell delivery device, where the quantity of cells may be provided in the internal device and injected into the tissue of the subject through the same injection attachment 100. For example, neither the cannula tip 121 nor the cannula 120 is altered between providing the quantity of cells into the internal volume and injecting the cells into the tissue of the subject.

[0126] In certain aspects, the present disclosure provides a method for delivering cell therapy, such as cell aggregate or single cell suspension therapy. The method can be used for a variety of cell therapies known in the art. Delivery can include a microsurgical procedure, such as an ophthalmic procedure, using a microinjection cannula or similar device. Delivery can include subretinal injection via slow, controlled infusion of the cell therapy. The cannula can be connected to a syringe or system that allows for a predetermined, uniform injection pressure (psi), e.g., 0-30 psi (e.g., greater than 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 psi, or 0-30, 2-28, 4-26, 6-24, 5-20, 15-25, 20-25, or 25-30 psi, or 0-15, 0-16, 0-17, 0-18, 0-19, or 0-20 psi), which can be controlled by a variable pressure-control foot pedal 250. The cells may be stored at a suitable temperature, such as 4°C.

[0127] In certain embodiments, the delivery device provides a mechanism for ocular delivery, such as subretinal administration, or a methodology for delivery of a therapeutic medium to a mammalian eye (e.g., the posterior segment of the eye), more particularly a human eye, and for treating and / or preventing ocular disorders and / or diseases, particularly retinal / choroidal disorders or diseases, via such subretinal administration of such therapeutic medium. Such methodologies provide a mechanism for treating a wide range of diseases and / or disorders of the mammalian eye, more particularly the human eye, more particularly diseases or disorders involving the posterior segment of the eye, such as retinal / choroidal disorders or diseases. Such treatment / prevention methodologies may also be used to treat / prevent several vision-threatening disorders or diseases of the mammalian eye, including, but not limited to, diseases of the retina, retinal pigment epithelium (RPE), and choroid. Such vision-threatening diseases include, for example, ocular neovascularization, ocular inflammation, and retinal degeneration. Specific examples of these disease states include diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy, age-related macular degeneration, retinal neovascularization, subretinal neovascularization; inflammatory disease rubeosis iridis, chronic posterior uveitis and panuveitis, neoplasms, retinoblastoma, pseudoretinal glioma, neovascular glaucoma; neovascularization following combined vitrectomy and lensectomy, vascular diseases such as retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, optic nerve neovascularization, diabetic macular edema, cystic macular edema, macular edema, retinitis pigmentosa, retinal vein occlusion, proliferative vitreoretinopathy, vascularized striatum, and retinal artery occlusion, as well as neovascularization resulting from ocular penetration or injury. The method can also be used to treat ocular symptoms resulting from diseases or conditions that have both ocular and non-ocular symptoms.

[0128] In some embodiments, preparation and delivery of the cell composition includes thawing and resuspending the cell composition in a vial (e.g., a 50 mL conical tube). The cell composition may be formulated in a buffer as described herein. The cell composition may be centrifuged, for example, one, two, three, four, five, or more times (e.g., in the formulation buffer). For clinical applications, a bulk dose of the cell composition can be prepared in the formulation buffer (e.g., 25-50, 30-75, 40-80, 50-90, or 60-100 μL of buffer). At this stage, the cell composition may be dissociated and counted. Next, a master dose can be prepared to reach a target concentration of the cell composition. Finally, the dose can be aliquoted (e.g., 50 μL) into an appropriate vial. For animal studies, such as rodent studies, the cell composition can be dissociated and counted, and then the remaining volume can be centrifuged. A master dose can be prepared by aspirating the buffer, resuspending the pellet with the remaining supernatant, measuring the volume, and adding formulation buffer to achieve the target concentration. A dose (eg, 15 μL) can then be dispensed.

[0129] The device may be used for transplants such as cell rescue therapy or total tissue replacement therapy. Certain embodiments may provide for the use of the device to enhance ocular tissue maintenance and repair for any condition requiring ocular tissue maintenance and repair, including retinal degeneration or significant damage. Retinal degeneration may be associated with age-related macular degeneration (AMD), hereditary macular degeneration, Stargardt's macular dystrophy, Best's disease, choroideremia, inherited retinal degenerations (including retinitis pigmentosa, cone / rod and rod / cone dystrophy), diabetic retinopathy, retinal vascular disease, damage caused by retinopathy of prematurity (ROP), viral infections of the eye, and other retinal / ocular diseases or injuries / trauma.

[0130] In another aspect, the present disclosure provides a method of treating an individual in need thereof, comprising transplanting a composition comprising cells, such as PRP cells, into the individual. The composition may be administered to the eye, e.g., the subretinal space. Such individuals may have inherited macular degeneration or retinal degeneration, such as retinitis pigmentosa, cone / rod or cone / cone dystrophy, Stargardt disease, Best disease, choroideremia, retinal dysplasia, retinal degeneration, diabetic retinopathy, congenital retinal dystrophy, Leber's congenital amaurosis, retinal detachment, damage caused by retinopathy of prematurity (ROP), or other retinal trauma or damage.

[0131] The devices and cells described herein can be used in the manufacture of pharmaceuticals to treat conditions in patients in need thereof. The cells can be previously cryopreserved. In certain aspects, the disclosed photoreceptors, because they are derived from iPSCs, can be used to provide "personalized medicine" to patients with ocular diseases. In some embodiments, somatic cells obtained from a patient can be genetically engineered to correct disease-causing mutations and differentiated into PRP. Alternatively, iPSCs generated from healthy donors or HLA-homozygous "super donors" can be used.

[0132] Various ocular conditions can be treated or prevented by the present methods and compositions. Conditions include retinal diseases or disorders commonly associated with retinal dysfunction or degradation, retinal damage, and / or loss of retinal pigment epithelium and / or photoreceptors. Conditions that can be treated include, but are not limited to, retinal degenerative diseases, such as Stargardt's macular dystrophy, retinitis pigmentosa, rod / cone and cone / rod dystrophies, macular degeneration (e.g., age-related macular degeneration, myopic macular degeneration, or other acquired or hereditary macular degenerations), retinal damage caused by retinopathy of prematurity (ROP), and diabetic retinopathy. Additional conditions include Leber's congenital amaurosis, hereditary or acquired macular degeneration or retinal degeneration, Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophies, other dystrophies of photoreceptor cells, and retinal damage resulting from damage caused by any one of light, laser, inflammatory, infectious, radiation, neovascular, or traumatic injury. In certain embodiments, a method for treating or preventing a condition characterized by retinal degeneration is provided, comprising administering, via the device, to a subject in need of such treatment or prevention, an effective amount of a composition comprising PRP.

[0133] In some embodiments, the cell delivery device and its method of use provide a mechanism for subretinal administration or delivery of cells to the posterior segment of a mammalian eye, more particularly a human eye, and a methodology for treating and / or preventing ocular disorders and / or diseases, particularly retinal / choroidal disorders or diseases, through such subretinal administration of such therapeutic media. Such methodology provides a mechanism for treating a wide range of diseases and / or disorders of the mammalian eye, more particularly a human eye, more particularly diseases or disorders involving the posterior segment of the eye, such as retinal / choroidal disorders or diseases. Such treatment / prevention methodology can also be used to treat / prevent several vision-threatening disorders or diseases of the mammalian eye, including, but not limited to, diseases of the retina, retinal pigment epithelium (RPE), and choroid. Such vision-threatening diseases include, for example, ocular neovascularization, ocular inflammation, and retinal degeneration. Specific examples of these disease states include diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy, age-related macular degeneration, retinal neovascularization, subretinal neovascularization; inflammatory disease rubeosis iridis, chronic posterior uveitis and panuveitis, neoplasms, retinoblastoma, pseudoretinal glioma, neovascular glaucoma; neovascularization following combined vitrectomy and lensectomy, vascular diseases such as retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, optic nerve neovascularization, diabetic macular edema, cystic macular edema, macular edema, retinitis pigmentosa, retinal vein occlusion, proliferative vitreoretinopathy, vascularized striatum, and retinal artery occlusion, as well as neovascularization resulting from ocular penetration or injury. The method can also be used to treat ocular symptoms resulting from diseases or conditions that have both ocular and non-ocular symptoms.

[0134] The delivery method can be used for cell transplantation for various cell therapies, such as the treatment of peripheral arterial disease, myocardial infarction, stroke, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). The cells can be pluripotent stem cells, stem cells such as mesenchymal stem cells (MSCs), bone marrow cells or neural progenitor cells (NPCs), cardiac progenitor cells, or iPSC-derived cells such as neurons, stromal cells, fibroblasts, endothelial cells, epithelial cells, etc. The device and method can be used to transplant cells into any soft tissue, including, but not limited to, the eye, heart, kidney, liver, tumor, or muscle. Cell doses can range from hundreds to millions of cells.

[0135] V. Cell composition The terms "cell population," "cell composition," "cell therapy composition," or "composition of cells" are typically used interchangeably herein to refer to a group of cells of a common type. A cell population may be derived from a common progenitor cell or may contain multiple cell types. An "enriched" cell population refers to a cell population derived from a starting cell population that contains a higher percentage of a particular cell type than the percentage of that cell type in the starting population (e.g., an unfractionated heterogeneous cell population). A cell population is enriched for one or more cell types and depleted for one or more cell types.

[0136] The cell composition can comprise single cell suspension or cell aggregate. The cell composition can be autologous or allogeneic. The cell composition can comprise or be derived from PSC, for example, iPSC or hESC. The cell composition can comprise one cell type (i.e., single cell) or two or more cell types (i.e., multicellular). In some embodiments, the term "cell" used herein refers to the cell composition. In some embodiments, the cell composition can comprise organoid or can be total tissue replacement.

[0137] In some aspects, the cell aggregates may comprise cell clusters. The cell clusters may comprise at least two cells. In some embodiments, the cell clusters comprise more than three, more than four, more than five, more than ten, more than fifteen, more than twenty, more than twenty-five, or more than thirty cells. In some embodiments, the cell clusters comprise at least three, at least four, at least five, at least ten, at least fifteen, at least twenty, at least twenty-five, or at least thirty cells. A single cell may be approximately 9 μm in diameter. In some embodiments, a single cell is approximately 7 μm, approximately 7.5 μm, approximately 8 μm, approximately 8.5 μm, approximately 9 μm, approximately 9.5 μm, approximately 10 μm, approximately 10.5 μm, or approximately 11 μm in diameter. The cell composition may comprise a distribution of cell clusters varying in size therein.

[0138] In some embodiments, it includes pluripotent stem cells (PSCs) or cells derived therefrom. PSCs can include embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), which have the ability to differentiate into several cell types, which can also be used for drug testing and disease research and treatment. PSCs can differentiate into any of the 216 cell types found in adult organisms, such as neurons, cardiomyocytes, smooth muscle cells, bone cells, hepatocytes, keratinocytes, insulin-producing cells, hematopoietic cells, and endothelial cells. In certain embodiments, the PSC-derived cells can include one or more of the 216 cell types. In some embodiments, the transplanted cell composition comprises engineered stem cell-derived tissues or organs composed of multiple cell types. The primary cell types used, derived from endoderm, include hepatocytes and insulin-producing cells. Mesoderm progenitors obtained from ESCs and iPSCs include cardiomyocytes, endothelial cells, and hematopoietic cells. These cell types can be used to treat ischemic heart disease, repair ischemic tissue, and obtain all types of blood cells, respectively. Cells differentiated along ectodermal lineages include cells of the epidermis, external sensory organs and the central and peripheral nervous systems, e.g., functional neurons that can be used to treat neurodegenerative diseases such as acute spinal cord injury.

[0139] In some embodiments, the cell compositions can be genetically engineered to knock out and / or knock in genes, which can be done by a variety of genome editing approaches, including CRISPR technology, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) technology.

[0140] The cell therapy composition may also include other agents. For example, the composition may include agents that further protect or stabilize the transplanted cells. In certain embodiments, the composition includes vitamins, minerals, antioxidants, osmoprotectants, viscosity enhancing agents, coenzymes, membrane stabilizers, lipids, carbohydrates, hormones, growth factors, anti-inflammatory agents, polynucleotides, proteins, peptides, alcohols, organic acids, and small organic molecules, among others.

[0141] The cell composition may contain pharmaceutically acceptable excipients, including any solvent, dispersion medium, diluent, or other suitable for the particular formulation desired, as described herein. These include liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, and lubricants. Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) may be used to formulate pharmaceutical compositions. Various excipients that can be used and known techniques for preparing pharmaceutical compositions are disclosed. Any conventional excipient may cause some undesired biological effect by the substance or its derivatives or adversely interact with any other components of the pharmaceutical composition. Therefore, unless otherwise used, its use is intended to be within the scope of the present invention.

[0142] In some embodiments, the pharmaceutically acceptable excipient is at least 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0143] Pharmaceutically acceptable excipients used in the production of cell therapy compositions include, but are not limited to, inert diluents, dispersing agents, surfactants and / or emulsifiers, disintegrants, preservatives, buffers, and lubricants. Such excipients may optionally be included in the formulations of the present invention. Excipients such as coloring agents may be present in the composition at the discretion of the formulator. Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, and kaolin. Examples include mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and the like, as well as combinations thereof.

[0144] Exemplary buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium glucoseptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monopotassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, phosphoric acid, sodium bicarbonate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and the like, and combinations thereof.

[0145] In further embodiments, cell transplantation kits are provided herein. The kits may include buffers for cell washing, devices for cell washing, cells, syringes, needles, cups, containers, alcohol swabs, anesthetics, antibiotics, antioxidants, vitamins, lipids, carbohydrates, hormones, and growth factors, etc. In certain embodiments, the kits include cannulas with funnel-shaped hubs, tubing, and pneumatically driven syringes, such as the MICRODOSE™ syringe. In certain embodiments, the kit components are sterilized and packaged for convenient use by surgeons or other healthcare professionals or during the manufacturing process. The kits may also include instructions for using the cannulas and other agents in the transplantation procedure. The kits may provide the necessary components for a single use. The kits may also include packaging materials and information required by government regulators regulating pharmaceuticals and / or medical devices.

[0146] VI. Eye cells In some embodiments, retinal progenitor cells (RPE), photoreceptors, and / or photoreceptor progenitor cells are delivered by the devices provided herein. Cells in the retina that are directly sensitive to light are photoreceptor cells. Photoreceptors are light-sensitive neurons in the outer part of the retina and can be either rods or cones. In the process of phototransduction, photoreceptor cells convert incident light energy focused by the cornea and lens into electrical signals that are ultimately sent to the brain via the optic nerve. Vertebrates have two types of photoreceptor cells, including cones and rods. Cones are adapted to detect fine detail, central and color vision and function well in bright light. Rods are responsible for peripheral and dim light vision. Neural signals from rods and cones are processed by other neurons in the retina.

[0147] Photoreceptors can express markers such as OTX2, CRX, PRDM1 (BLIMP1), NEUROD1, RCVRN, TUBB3, and L1CAM (CD171). Photoreceptors express several proteins that can serve as markers for detection using methodologies such as immunocytochemistry, Western blot analysis, flow cytometry, or enzyme-linked immunosorbent assay (ELISA). For example, one characteristic photoreceptor marker is RCVRN. Photoreceptors may not express (at any detectable level) the embryonic stem cell markers OCT-4, NANOG, or REX-1. Specifically, the expression of these genes is approximately 100-1000-fold lower in photoreceptors than in ES cells or iPSC cells, as assessed by quantitative RT-PCR.

[0148] Photoreceptor markers can be detected at the mRNA level by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, microarrays, or RNA sequencing, including, for example, single-cell RNA sequencing dot blot hybridization analysis using sequence-specific primers in standard amplification methods using publicly available sequence data (GENBANK®). Expression of a tissue-specific marker detected at the protein or mRNA level is considered positive if the level is at least or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold higher than that of control cells, e.g., undifferentiated pluripotent stem cells or other unrelated cell types, and more particularly, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more.

[0149] Dysfunction, damage, and loss of photoreceptor cells are factors in many ocular diseases and disorders, including age-related macular degeneration (AMD), hereditary macular degeneration (including Best disease, Stargardt disease, and choroideremia), retinitis pigmentosa, and other forms of inherited retinal diseases, as well as acquired retinal dysfunction, disease, and injury. A potential treatment for such diseases is the transplantation of PRP and / or PR into the retina of patients in need of such treatment. It is speculated that supplementation of PRP and / or PR by such transplantation may slow, stop, or reverse degradation, improve retinal function, and prevent blindness resulting from such conditions. However, obtaining PRP and / or PR directly from human donors and embryos is difficult.

[0150] The retinal pigment epithelium (RPE) acts as part of the barrier between the bloodstream and the retina and interacts closely with photoreceptors to maintain visual function and choroidal blood supply. It is composed of a single layer of hexagonal cells densely packed with melanin granules. The main functions of specialized RPE cells include transporting nutrients such as glucose, retinol, and fatty acids from the blood to photoreceptors, transporting water, metabolic end products, and ions from the subretinal space to the blood, absorbing light and protecting against photooxidation, re-isomerizing all-trans-retinol to 11-cis-retinal, phagocytosis of shed photoreceptor membranes, and secreting various essential factors for the structural integrity of the retina.

[0151] Mature retinal pigment epithelium expresses markers such as cellular retinaldehyde-binding protein (CRALBP), RPE65, Best vitelliform macular dystrophy gene (VMD2), and pigment epithelium-derived factor (PEDF). Dysfunction of the retinal pigment epithelium is associated with several visual changes, such as retinal pigment epithelial detachment, dysplasia, atrophy, retinopathy, retinitis pigmentosa, macular dystrophy, or degeneration, including age-related macular degeneration.

[0152] Mature retinal pigment epithelial (RPE) cells can be characterized based on their pigmentation, epithelial morphology, and apical-basal polarity. Differentiated RPE cells can be visually recognized by their cobblestone morphology and initial appearance of pigment. In addition, differentiated RPE cell layers possess a transepithelial resistance (TER), generate a transepithelial potential (TEP) across the apical-to-basal monolayer (TER > 100 ohms·cm2; TEP > 2 mV), transport fluid, lactate, and CO2, and regulate polarized cytokine secretion.

[0153] RPE cells express several proteins that can serve as markers for detecting their identity and maturation state using methodologies such as immunocytochemistry, Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). For example, RPE-specific markers include cellular retinaldehyde-binding protein (CRALBP), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TYRP-1), retinal pigment epithelium-specific 65 kDa protein (RPE65), premelanosome protein (PMEL17), bestrophin 1 (BEST1), and c-mer proto-oncogene tyrosine kinase (MERTK). At the same time, RPE cells do not express (at any detectable level) the embryonic stem cell markers Oct-4, nanog, or Rex-1. Specifically, the expression of these genes is approximately 100-1000-fold lower in RPE cells than in ES or iPSC cells, as assessed by quantitative RT-PCR.

[0154] RPE cell markers can be detected at the mRNA level by standard amplification methods, such as reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot blot hybridization analysis using sequence-specific primers, using published sequence data (GENBANK®). Expression of a tissue-specific marker detected at the protein or mRNA level is considered positive if the level is at least or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold higher than that of control cells, such as undifferentiated pluripotent stem cells or other unrelated cell types, and more particularly, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more.

[0155] Dysfunction, damage, and loss of RPE cells are contributing factors to many ocular diseases and disorders, including, but not limited to, age-related macular degeneration (AMD), hereditary macular degeneration (including Best disease, Stargardt disease, and choroideremia), and other forms of inherited retinal disease, as well as acquired retinal dysfunction, disease, and damage, including, but not limited to, RPE rupture / tear. A potential treatment for such diseases is the transplantation of RPE cells into the subretinal space of patients in need of such treatment. It is speculated that replenishing RPE cells by their transplantation may slow, stop, or reverse degradation, improve retinal function, and prevent blindness resulting from such conditions. However, obtaining RPE cells directly from human donors and embryos is challenging.

[0156] In some aspects, RPE cells are generated from iPSCs, such as by the methods disclosed in International Application Nos. PCT / US2016 / 050543 and PCT / US2016 / 050554.

[0157] In some embodiments, methods are provided for generating photoreceptors from essentially single cell suspensions of PSCs, such as human iPSCs. In some embodiments, the PSCs are cultured to preconfluence. In certain aspects, the PSCs are dissociated by incubation with a cell dissociation solution or enzyme, exemplified by Versene, trypsin, ACCUTASE™, or TRYPLE™. PSCs can also be dissociated into essentially single cell suspensions by pipetting.

[0158] Additionally, blebbistatin (e.g., approximately 2.5 μM) can be added to the culture medium to prevent cells from adhering to the culture vessel while increasing PSC survival after dissociation into single cells. Alternatively, a ROCK inhibitor can be used instead of blebbistatin to increase PSC survival after dissociation into single cells.

[0159] Once a single cell suspension of PSCs is obtained, the cells are generally seeded into an appropriate culture vessel, such as a tissue culture plate, e.g., a flask, a multi-layer flask, a 6-well, 12-well, 24-well, 96-well, or 10 cm plate. The culture vessel used to culture the cells may include, but is not limited to, a flask, a tissue culture flask, a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multi-plate, a multiwell plate, a microslide, a chamber slide, a tube, a tray, a CELLSTACK® chamber, a culture bag, and a roller bottle, so long as stem cells can be cultured therein. Cells may be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range of volumes derivable therein, depending on the needs of the culture. In certain embodiments, the culture vessel may be a bioreactor, which may refer to any ex vivo device or system that supports a biologically active environment in which cells can grow. The bioreactor can have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0160] In certain embodiments, PSCs, such as iPSCs, are plated at a cell density appropriate for efficient differentiation. Generally, cells are plated at a density of about 1,000 to about 75,000 cells / cm. 2 , for example, about 5,000 to about 40,000 cells / cm 2In a 6-well plate, the cells may be seeded at a cell density of about 50,000 to about 400,000 cells per well. In an exemplary method, the cells are seeded at a cell density of about 100,000, about 150,000, about 200,000, about 250,000, about 300,000, or about 350,000 cells per well, e.g., about 50,000 cells per well.

[0161] PSCs, such as iPSCs, are typically cultured on culture plates coated with one or more cell adhesion proteins to promote cell attachment while maintaining cell viability. For example, preferred cell adhesion proteins include extracellular matrix proteins, such as vitronectin, laminin, collagen, and / or fibronectin, which can be used to coat culture surfaces as a means of providing a solid support for pluripotent cell growth. The term "extracellular matrix (ECM)" is art-recognized. Its components may include, but are not limited to, one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Other ECM components may include synthetic peptides for adhesion (e.g., RGD or IKVAV motifs), synthetic hydrogels (e.g., PEG, PLGA, etc.), or natural hydrogels such as alginate. In an exemplary method, PSCs are grown on vitronectin-coated culture plates. In some embodiments, the cell adhesion protein is a human protein.

[0162] Extracellular matrix proteins can be of natural origin and purified from human or animal tissue, or alternatively, ECM proteins can be genetically engineered recombinant proteins or natural synthetic products. ECM proteins can be in the form of natural or engineered whole proteins or peptide fragments. Examples of ECM proteins that may be useful in matrices for cell culture include laminin, collagen I, collagen IV, fibronectin, and vitronectin. In some embodiments, the matrix composition is xeno-free. For example, a xeno-free matrix for culturing human cells can use matrix components of human origin and exclude any non-human animal components.

[0163] In some aspects, the total protein concentration in the matrix composition can be about 1 ng / mL to about 1 mg / mL. In some preferred embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0164] Cells, such as photoreceptors or PSCs, can be cultured with nutrients essential for supporting the growth of each specific cell population. Generally, cells are cultured in a growth medium containing a carbon source, a nitrogen source, and a buffer to maintain pH. The medium may also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, pH indicators, and inorganic salts. An exemplary growth medium contains a minimal essential medium, such as Dulbecco's Modified Eagle's Medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance stem cell growth. Examples of minimal essential medium include, but are not limited to, Minimal Essential Medium Eagle's (MEM) Alpha Medium, Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 Medium, 199 Medium, and F12 Medium. Additionally, the minimal essential medium may be supplemented with additives such as horse, calf, or fetal bovine serum. Alternatively, the medium may be serum-free. In other cases, the growth medium may contain "Knockout serum replacement," referred to herein as a serum-free formulation optimized to grow and maintain undifferentiated cells, such as stem cells, in culture. KNOCKOUT™ serum replacement is disclosed, for example, in U.S. Patent Application Publication No. 2002 / 0076747, which is incorporated herein by reference. Preferably, PSCs are cultured in a fully defined, feeder-free medium.

[0165] Thus, single-cell PSCs are generally cultured in a completely defined culture medium after plating. In certain embodiments, about 18-24 hours after plating, the medium is aspirated and fresh medium, such as E8™ medium, is added to the culture. In certain embodiments, single-cell PSCs are cultured in a completely defined culture medium for about 1, 2, or 3 days after plating. Preferably, single-cell PSCs are cultured in a completely defined culture medium for about 2 days before proceeding with the differentiation process.

[0166] In some embodiments, the medium may or may not contain any substitute for serum. Serum substitutes may include materials that suitably contain albumin (e.g., lipid-enriched albumin, albumin substitutes such as recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum substitutes may be prepared, for example, by the methods disclosed in WO 98 / 30679. Alternatively, any commercially available material may be used for greater convenience. Commercially available materials include KNOCKOUT™ Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and GLUTAMAX™ (Gibco).

[0167] Other culture conditions can be defined as appropriate. For example, the culture temperature can be about 30 to 40°C, for example, at least about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not limited thereto. In one embodiment, cells are cultured at 37°C. The CO2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen partial pressure can be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any range derivable therein.

[0168] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, understand that many modifications can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. [Example]

[0169] Example 1 – Delivery of photoreceptor precursor cell aggregates We conducted a study to develop a dose delivery technique for both large animal and clinical settings. Allogeneic human induced pluripotent stem cell (iPSC)-derived photoreceptor progenitor cell (iPRP) aggregates were developed as a cell-based RP replacement therapy. After dose preparation (REP-00798), iPRP aggregates were implanted using Alcon's Constellation Vision System [1], an ophthalmic microsurgical system. A MICRODOSE™ Injection Kit 1 mL syringe (MedOne Surgical, Inc., no. 3275) [2] was attached to the CONSTELLATION® Vision System (Alcon) via tubing from a viscous fluid control (VFC) pack (no. 8065750957). We conducted a study to identify the most appropriate dose loading and delivery technique, including the creation and testing of novel cannula hub geometries for the delivery of cell products, such as aggregated cell products.

[0170] Dose Formulation. To prepare iPRP aggregates for transplantation, cells were thawed and resuspended in 0.2% HSA buffered BSS unless otherwise noted. Multiple spin and wash steps followed, along with dissociation with 10x TrypLE and removal of samples for cell counting. Viable cell concentrations were used to produce dose formulations (Figure 3).

[0171] Dose Analysis. For bench testing, the dose was loaded into a 1 mL syringe (BD #309628 or MedOne Surgical, Inc., #3275) through an 18 G needle and then expelled through a 31 G (MedOne #3218) or 33 G (MedOne #3262) cannula (single-pass method), or loaded into a syringe through a 31 G (MedOne #3218) or 33 G (MedOne #3262) cannula and then expelled through the same cannula (dual-pass method). Pressure to draw and expel the dose was generated manually by a plunger (BD #309628) or by attaching a 1 mL syringe (MedOne Surgical, Inc., #3275) to a CONSTELLATION® Vision System via a viscous fluid control (VFC) pack (#8065750957).

[0172] Upon release, aggregates were enzymatically dissociated in 10x TrypLE for 30 minutes, quenched in DMEM / F12+B27+Benzonase (iPRP quenching medium), triturated, and counted to analyze the number of recovered cells. Alternatively, to approximate the integrity of the aggregates, aggregates were transferred to a Multisizer 4 and passed through a 280 μm opening.

[0173] Overview of iPRP aggregate device construction. To deliver iPRP aggregates to the subretinal space of large animals or humans in the clinic, a CONSTELLATION® Vision System (Alcon, Figure 4) [1] was used to load and inject each volume at controlled pressure. A MICRODOSE™ Injection Kit 1 mL syringe (MedOne Surgical, Inc., number 3275) [2] was attached to the CONSTELLATION® Vision System via tubing from a viscous fluid control (VFC) pack (number 8065750957). An injection attachment 100 (MedOne Polytip Cannula, 25G / 33G) was connected to the syringe, through which a volume of 50 μL of cells was delivered into the subretinal space via a predetermined, uniform injection pressure (psi) controlled by a foot pedal.

[0174] Comparison of 31G vs. 33G cannulas. The integrity of iPRP aggregates can be adversely affected by how they are handled. Therefore, larger diameter cannulas may be used to reduce shear forces during dose loading and injection. However, because the cannula must pass through a retinotomy to deliver the dose posterior to the retina, a smaller cannula may be required to minimize the amount of damage to the retina. Additionally, the larger the retinotomy (to allow for a smaller gauge cannula), the wider the hole created in the retina, increasing the incidence of reflux of injected iPRP aggregates from the bleb and subsequently reducing product delivery. Historically, 31Ga cannulas have been used, but due to their potentially beneficial impact on surgical technique, this study investigated whether a smaller diameter cannula (33G) adversely affected the aggregate product.

[0175] To test the effect of cannula gauge on aggregate integrity, iPRP aggregates were thawed and prepared. 200 μL of the final aggregate product formulation was manually loaded into a syringe through an 18G needle. The needle was then replaced with either a MedOne 31G (item 3218) or a 33G POLYTIP® cannula (item 3262). Trapped air was removed from the syringe and cannula, and the unused portion of the dose was expelled until the plunger reached the 50 μL gradation (clinical dose). The 50 μL dose was then expelled, and aggregate size was analyzed using a Multisizer (280 μm opening). A gate was retracted from approximately 17 μm to 168 μm to capture aggregates, and the percentage of the product volume that fell within that gate was measured. Additionally, the median aggregate diameter within that gate was calculated. Based on these results, the use of a 33G cannula should be comparable to a 31G POLYTIP® cannula in terms of aggregate integrity, as no significant differences in aggregate quantity or median diameter were observed. Because the effect of cannula diameter on aggregate integrity and surgical benefit is negligible when using smaller cannulas, further studies were performed with a 33G POLYTIP® cannula (number 3262).

[0176] Investigation of Variable Surgical Dose Delivery. iPRP is composed of aggregates and therefore settles rapidly from formulation buffers (REP-00798). This not only affects the dose formulation but can also cause problems during dose delivery. Therefore, a study was performed to determine whether settling of the product in the syringe could be a cause of inaccurate dosing.

[0177] Historically, in vitro experiments during this period used a manual 1 mL syringe attached to an 18G needle during loading (200 μL), which was then removed to release cells (50 μL) through a 33G cannula. The 50 μL injection of iPRP aggregates was then dissociated in 10× TrypLE and counted. Note that the experiments presented here were originally intended to investigate various formulation vehicles (BSS Plus, BSS Part I only, BSS Plus with Benzonase, BSS Plus with 0.2% HSA, BSS Plus with 0.2% HSA and Benzonase, standard BSS, and RMN), but the data were combined for this study due to the lack of clear vehicle effects. Results are presented as a percent of expected cell recovery (e.g., 30-100%, 30-50%, 40-60%, 40-90%, 50-70%, 60-80%, 70-90%, or 80-100%), which was calculated by dividing the actual viable cells recovered by the target dose (Figure 6, blue circle). After eight experiments across four lots (iPRP0045 20200727-CD133_60, iPRP0047A, iPRP0047B, and iPRP0049A) with a total of 33 individual injections, doses fell within approximately 40-90% of the expected range.

[0178] At approximately this point, a video of a mock surgery using this cell product performed by the surgeon and surgical assistant was provided. From this experiment, very few cells were recovered after injection. Review of the video indicated that the surgical process was slower. Additionally and importantly, not only was the time increased, but the tip of the cannula was oriented upward for the majority of the time, which may have prevented the aggregates from settling toward the syringe plunger and being injected. To test this hypothesis, three doses of iPRP0047A were generated (one for each test condition). A control was performed in the same manner as the previous eight experiments, in which the dose was drawn up and injected rapidly (FCDI standard; Figure 6, dashed line). Specifically, in the FCDI standard, the cell aggregate composition is resuspended in BSS with 0.2% HSA. The Constellation Vision system was attached to the tubing, viscous fluid control pack, and microdose injection kit syringe. The syringe and cannula were loaded with buffer with the cannula facing up, and the foot pedal was depressed to purge excess air and BSA+HSA. The syringe was inserted vertically into a clamp on a ring stand with the cannula tip facing down. The Constellation system was set to extract a 50 μL dose into the syringe and cannula and allowed to settle for approximately 5 minutes. The Constellation system was then set to inject at 10 PSI when the foot pedal was depressed. For the second dose, the scientist reflected any changes in syringe orientation made by the surgeon on video from the period of dose uptake by injection (surgeon's orientation; Figure 6, rectangle). Specifically, the surgeon's orientation included holding the dose perpendicular to the ground during settling and at an angle A of approximately 45 degrees, as expected for injection, as shown in Figure 17B. To determine whether time in the syringe reduced dose recovery, in the third condition, the syringe was loaded and then held for the same amount of time as the surgeon manipulated the dose (approximately 5 minutes), but was held parallel to the ground and rolled back and forth between the scientist's fingers for the purpose of keeping the aggregates in suspension (Figure 6, diamond).

[0179] Upon collection, all iPRP aggregate injection doses were dissociated in 10x TrypLE and counted to calculate the expected percent cell recovery. The surgeon-directed dose stood out with a recovery rate of less than 20% (injection #35; Figure 6, pink rectangle), whereas the FCDI standard dose (injection #34; Figure 6, green dashed line) and the 5-minute rolling dose (injection #36; Figure 6, light blue diamond) had typical recovery rates. These findings highlight the need to either 1) thoroughly train surgeons and surgeon assistants to maintain aggregates in suspension, or 2) modify the delivery approach.

[0180] Flicking the loaded syringe is sufficient to maintain the aggregate suspension. With the knowledge that iPRP aggregates may shed from the formulation buffer once loaded into the syringe, potentially resulting in an inaccurate injected dose, we sought to identify a method to maintain proper aggregate distribution within the syringe. Using lot iPRP0046C, three separate experiments were performed. The dose was prepared so that 2 million cells were expected to be delivered from the injection. Using a Constellation syringe, 200 μL was loaded into the syringe through an 18G needle. After replacing the 18G needle with a 33G cannula (MedOne #3262), air was purged and the excess volume was expelled so that the plunger reached the 50 μL gradient. Typically, at this step in the protocol, the dose is in the device within 2 minutes before injection. To simulate a potential "worst-case scenario," the syringe was placed on its side for 5 minutes, a time during which aggregates visibly settled on the side of the syringe. After this waiting period, the dose was either immediately released (no mixing) or first mixed by vortexing or flicking the syringe. A total of 14 injections were performed across these three experiments. As expected, mixing before releasing the dose from the syringe resulted in significant recovery (Figure 7). Based on these results, flicking was determined to be sufficient when redistributing the dose.

[0181] We take advantage of aggregate sedimentation by using the injection attachment 100. Flicking the syringe loaded with iPRP aggregates redistributes the settled aggregates, providing a mixed formulation and reliable injection (Figure 7). However, despite these modifications, concerns regarding variability in surgical injections remained, so we tested various dose delivery methods, including intentionally settling the aggregates within the hub of the cannula.

[0182] We hypothesized that loading a 50 μL dose into a syringe and allowing all aggregates to settle vertically into the cannula hub would ensure that all aggregates would be released initially during dose injection, reducing the likelihood of them becoming trapped along the side of the syringe or against the plunger due to settling and improper resuspension technique. Therefore, we next tested whether this new settling method would improve aggregate recovery over the previous suspension method. In this experiment, iPRP aggregates were prepared for a "resuspension" dose. As in the previous experiment, a 200 μL dose was manually drawn into a syringe through an 18G needle. The 18G needle was then replaced with a 33G cannula (MedOne #3262) to remove air and excess volume, leaving a 50 μL dose within the device. This 50 μL dose was then injected into a tube for analysis. However, for vertical sedimentation studies, a 33G cannula was attached to a syringe, and both were loaded with vehicle (BSS + HSA) to ensure no air was trapped in the cannula or syringe. Once the air was purged, the plunger was pushed back to 0 μL. Then, with the device held vertically (cannula pointing downward), a 50 μL dose was drawn through the cannula to initiate a 5-minute wait period. After the incubation period, this 50 μL dose was also expelled into a tube for downstream analysis. The cells were then dissociated and counted to determine the delivered dose. Unfortunately, while the suspension method achieved the expected recovery of approximately 2 million cells, the syringe that sedimented the cells injected less than 25% of the expected dose (Figure 8A).

[0183] The reason for this lack of cell recovery became apparent upon closer inspection of the cannula hub, which showed the space where cells were trapped as designed (Figure 8B, arrow). A ring of white aggregates was visible within the cannula hub 301 even after the syringe plunger was fully depressed (Figure 8C, arrow).

[0184] Therefore, we developed an injection attachment 100 with a hub shaped like a funnel (FIG. 8D), which allows for less restricted space for aggregate entrapment compared to off-the-shelf cannulas (MedOne number 3262, FIG. 8B). We then conducted experiments with this new cannula design, hoping that the sedimentation method could improve the consistency of injection doses compared to the suspension method.

[0185] Returning to the same experimental design as for Figure 8A, we compared a prefabricated 33G POLYTIP® cannula (number 3262) using the suspension method with the injection attachment 100 using the aggregation and sedimentation method. In the first experiment, iPRP0046C, a dose of iPRP aggregate was prepared and seven injections were performed using the injection attachment 100 according to the sedimentation method, while five injections were performed using the commercially available cannula according to the suspension method (Figure 8E). The following experiments were performed in exactly the same way with the same iPRP aggregate, using four injections using the injection attachment 100 and five injections using the prefabricated cannula (Figure 8F). Using the injection attachment 100 using the sedimentation method, the CVs were 9.2 and 8.4%, respectively, while the prefabricated cannula showed CVs of 29.2 and 23.4%, respectively. This reduced variability in the injection attachment 100 and sedimentation method eliminated the need to maintain the aggregates in suspension, subsequently alleviating concerns regarding proper surgical training to accurately deliver iPRP aggregates.

[0186] In the "resuspension" method, aggregates entered the cannula through an 18G needle and exited through a 33G cannula, whereas in the "sedimentation" method, aggregates exited through a 33G cannula. Because the integrity of aggregates can be affected by shear stress, the integrity of aggregates that had passed through the cannula once or twice was investigated.

[0187] The first experiment was conducted to test the effect of two cannula passes on aggregate integrity. iPRP aggregates were thawed and doses were prepared. To test two-passage through the cannula, a 200 μL dose was loaded and injected as described above for the suspension method, except that a 200 μL dose was loaded into the syringe through a 33-gauge cannula instead of an 18-gauge needle. After release, a 50 μL dose was analyzed using a Multisizer with a 280 μm aperture. Gates were drawn from approximately 17 μm to 168 μm to measure the percentage of product volume that fell within that gate and the median aggregate diameter within that gate. The median aggregate diameter was found to be approximately 40-60 μm, e.g., 40-45, 45-55, 50-55, or 50-60 μm. A slight decrease in both measurements occurred when the aggregates were passed twice through the 33-gauge cannula, but the effect was small enough to allow the sedimentation method to continue (Figure 9).

[0188] Dose Variation Calculation. For many dosing studies, multiple concentrations were tested. For this workflow, the product was thawed, washed, and then resuspended in a set volume of BSS+HSA that would ensure the bulk was intentionally more concentrated than the final target concentration. Samples were then removed from this bulk dose and dissociated to obtain cell counts. The bulk volume was measured and then distributed into multiple tubes and diluted in additional BSS+HSA to target the appropriate concentration. These volumes, called master doses, were then distributed into aliquot doses used for infusion (Figure 10).

[0189] To determine variability between master doses, 28 master doses from 10 experiments were compared using seven different iPRP aggregate product lots (iPRP0046C, iPRP0047B, iPRP0049C, iPRP0049D, iPRP0054F, iPRP0055A, iPRP0057). To standardize reporting, the % expected cell concentration (actual counted concentration divided by target concentration) was plotted. Results showed that these master doses contained, on average, 93% of the expected cell concentration (coefficient of variation 11.4%) (Figure 11). 80% of the doses were within ±16% of the target cell concentration, 90% were within ±23% of the target dose, and 100% were within ±28% of our doses. The minimum expected cell concentration was 73%, and the maximum expected cell concentration was 111% (Figure 11).

[0190] One source of error that may have contributed to the variability in these studies was the use of a single bulk formulation to prepare master doses of multiple concentrations. However, this variability was present in either clinical or large animal GLP studies because the protocols required only one master dose concentration. For these studies, the bulk was prepared, samples were removed for dissociation and cell counting, and the bulk concentration was calculated. However, instead of dividing the bulk into multiple tubes, all but 20 μL of the bulk was transferred to a new tube and diluted to the target concentration (master dose). This master dose was then divided into aliquot doses for injection (Figure 12).

[0191] Experiments producing a single master dose from a single bulk concentration resulted in lower variability than producing multiple master doses from bulk. Preparation of nine master doses from five different iPRP aggregate lots showed that the average master dose had 93.5% of the expected cell concentration (coefficient of variation 5%). 88% of the doses were within ±10% of our target cell concentration, with the minimum expected cell concentration being 84% and the maximum expected cell concentration being 99% (Figure 13).

[0192] Because aggregates settle rapidly, variability can be introduced when distributing the master dose into dispensed doses. In nine experiments using iPRP aggregates, 5 to 12 aliquots containing either 60 to 70 µL or 210 µL were dispensed (this volume was exceeded so that each aliquot could contain either 50 µL or 200 µL after sampling for counting, allowing for use in downstream experiments). A 5 µL sample was removed from each aliquot, dissociated, and counted to determine the concentration of the dispensed dose.

[0193] As mentioned above, the % expected cell concentration was used for this data because various dose concentrations were targeted in these experiments. Importantly, although there was variability between experiments, the dose-to-dose variability within experiments showed a coefficient of variation range of approximately 7.5% to 15.5%.

[0194] Dose Compensation Factor. Although a new device loading (50 μl dose through a cannula) technique with sedimentation within the injection attachment 100 resulted in improved accuracy, further studies were conducted to evaluate dosing accuracy. During multiple studies, it was observed that fewer injected cells were recovered than expected based on the calculated dose concentration. Therefore, a dose compensation factor was considered.

[0195] Doses ranging from 1.4 to 3.4 million cells were prepared and infused using Constellation over 11 experiments using six different lots of iPRP aggregates (iPRP0047B, iPRP0049C, iPRP0049D, iPRP0054F, iPRP0055A, and iPRP0057), for a total of 82 injections. Extrapolation of a linear fit line across all data points generated a best-fit equation of y = 0.9255x - 0.5777 (Figure 15A). Using this equation, it was calculated that doses of 1.7 and 2.8 million cells would need to be loaded to achieve target doses of 1 million (clinical) and 2 million (non-human primate) cells injected, respectively. For the 3 million and 4 million (clinical) cell doses, a linear fit line was extrapolated across all data points generated for 391 individual injections across two iPSC lines, eight iPRP lots (16 sublots) for all four doses, and a best-fit equation of y = 0.0348x - 0.1239 was determined (Figure 15B). Using this equation, it was calculated that doses of 4.5 million and 5.95 million cells would need to be loaded to achieve the target doses of 3 million and 4 million cells injected, respectively. The results of testing 1M, 2M, 3M, and 4M dose targets can be seen in Figure 15C.

[0196] The variability of infused clinical doses using 1.7 million cell compensation was tested in six experiments using three lots (iPRP0047B, iPRP0055A, and iPRP0057) for a total of 25 infusions. The mean dose was 1.04 million cells, with a coefficient of variation (CV) of 19%. 80% of the doses were within ±27% of the target 1 million dose, 90% were within ±33% of our dose, and 100% were within ±42% of the dose, with a minimum infused dose of 640,000 cells and a maximum infused dose of 1.41 million cells (Figure 15B). For a non-human primate target of 2 million cells, a calculated dose of 2.8 million cells was generated. Across 15 injections across three experiments and four lots (iPRP0047B, iPRP0054F, iPRP0055A, and iPRP0057), the mean dose was 2.1 million cells with a CV of 16%. 80% of the doses were within ±19% of our target 1 million dose, 90% were within ±23% of our dose, and 100% were within ±38% of our dose, with a minimum dose of 1.23 million cells injected and a maximum dose of 2.45 million cells injected (15C).

[0197] Observations and improvements for large animal (FIG. 16) and clinical iPRP aggregate dose delivery were described above. The data suggest that the implemented changes may result in more reliable dose delivery immediately after each injection.

[0198] The initial protocol required loading cells through an 18G needle, which then had to be exchanged for a 31G cannula before injection. This protocol worked, but could be improved. A 31G cannula was sufficient to deliver cells subretinal. However, it required a larger retinotomy, potentially increasing the potential for retinal damage and reflux of iPRP aggregates. Testing a smaller-diameter 33G cannula showed that increased shear stress on the aggregates had only a minimal effect on aggregate size distribution. Therefore, further studies were performed with a 33G cannula.

[0199] Subsequent testing showed that the aggregates could be passed through the cannula twice without consequential damage. Since no impact on aggregate integrity was observed, dose loading was switched to a 33G cannula instead of an 18G needle. The reduced handling steps not only made the process easier, but also reduced the chance of contamination and needle sticks.

[0200] This dual-pass method also allowed for overhaul of syringe handling prior to dose delivery. The single-pass method required loading a 200 μL dose into the syringe to facilitate removal of air from the syringe and cannula. Once the air was removed, the volume was dispensed until a 50 μL dose remained. To prevent aggregate settling, both the loading process and the time between loading and delivery had to be performed quickly with this method; even then, variations in the cells delivered during the procedure were observed. Therefore, we considered intentionally settling aggregates in hopes of injecting the most consistent dose.

[0201] While settling aggregates toward the cannula tip did not initially work due to the geometry of the cannula hub, the design of the injection attachment 100, using a new hub that acts more like a funnel, provided a much more consistent dose. This newly designed cannula allowed a 50 μl dose to be loaded through the cannula and allowed to settle toward its tip for 5 minutes before injection, eliminating the need to keep the aggregates in suspension before injection. Note that in addition to improved dose consistency, this method uses one-quarter the volume used by the original method (50 μL versus 200 μL).

[0202] The subject technology is exemplified according to various aspects, for example, as described below. Various examples of aspects of the subject technology are described as numbered clauses (1, 2, 3, etc.) for convenience. These are provided as examples and are not intended to limit the subject technology. Note that any of the dependent clauses can be combined in any combination into independent clauses, e.g., clause 1 or clause 18. Other clauses can be similarly set forth.

[0203] Clause 1. A method of cell transplantation comprising injecting cells into tissue of a subject using an injection attachment comprising a cannula having a funnel-shaped hub.

[0204] Clause 2. The method of clause 1, wherein the cannula hub is less than 30 mm in length.

[0205] Clause 3. The method of clause 1 or 2, wherein the cannula comprises a cannula tip of 30 gauge or less.

[0206] Clause 4. The method of clause 3, wherein the cannula tip is blunt.

[0207] Clause 5. The method of clause 3, wherein the cannula tip is sharp.

[0208] Clause 6. The method of clause 5, wherein the cannula tip is further defined as the cannula tip.

[0209] Clause 7. The method of clause 3, wherein the cannula tip is a 34, 33, 32, 31 or 30 gauge cannula tip.

[0210] Clause 8. The method of clause 3, wherein the cannula tip is a 33 gauge cannula tip.

[0211] Clause 9. The method of any of clauses 3-8, wherein the cells are loaded into the cannula by a 30 gauge cannula tip or smaller.

[0212] Clause 10. The method of any of clauses 3-8, wherein the cells are loaded into the cannula by a 33 gauge cannula tip.

[0213] Clause 11. The method of any of clauses 3 to 8, wherein the cells are loaded into a cannula and injected into the tissue of the subject through the tip of the same cannula.

[0214] Clause 12. The method of any of clauses 1-11, wherein the cannula tip is not altered between loading into the cannula and injecting into said tissue of the subject.

[0215] Clause 13. The method of any of clauses 1 to 12, wherein the cannula tip is fabricated from a flexible polyimide material or metal.

[0216] Clause 14. The method of any one of clauses 1 to 13, wherein the cannula tip is flexible.

[0217] Clause 15. The method of any of clauses 1 to 13, wherein the cannula tip is rigid.

[0218] Clause 16. The method of any of clauses 1 to 7, wherein the hub includes a one-way check valve.

[0219] Clause 17. The method of any one of clauses 1 to 16, wherein the hub includes a coupling mechanism.

[0220] Clause 18. The method of clause 17, wherein the coupling mechanism is a luer lock.

[0221] Clause 19. The method of clause 18, wherein the luer lock is coupled to a dosing mechanism.

[0222] Clause 20. The method of clause 19, wherein the administration mechanism is a syringe.

[0223] Clause 21. The method of clause 20, wherein the syringe is a microinjection syringe.

[0224] Clause 22. The method of clause 20, wherein the syringe is further connected to a tubing system.

[0225] Clause 23. The method of clause 22, wherein the piping system is connected to a pressure control system.

[0226] Clause 24. The method of any of clauses 1 to 23, wherein the cells are delivered from the injection attachment at a controlled pressure.

[0227] Clause 25. The method of any of clauses 1 to 24, wherein the cells are not redistributed prior to loading into the injection attachment.

[0228] Clause 26. The method of any of clauses 1 to 25, wherein the cells are redistributed prior to loading into the injection attachment.

[0229] Clause 27. The method of Clause 26, wherein the cells are redistributed by vortexing or manual agitation.

[0230] Clause 28. The method of any of clauses 1 to 27, wherein the cells are injected into the eye of said subject.

[0231] Clause 29. The method of clause 28, wherein the cells are injected subretinal.

[0232] Clause 30. The method of any of clauses 1-29, wherein the cells are further defined as cell aggregates.

[0233] Clause 31. The method of any of clauses 1 to 29, wherein the cell is further defined as a single cell.

[0234] Clause 32. The method of any of clauses 1 to 31, wherein the cells are in a formulation buffer.

[0235] Clause 33. The method of Clause 32, wherein the formulation buffer is a balanced salt solution.

[0236] Clause 34. The method of Clause 33, wherein the balanced salt solution further comprises benzonase and / or human serum albumin.

[0237] Clause 35. The method of clause 30, wherein the cell aggregates are not in suspension when injected into the tissue of said subject.

[0238] Clause 36. The method of clause 35, wherein the cells reside within the cannula for at least 5 minutes between loading and injection to allow aggregate sedimentation within the cannula hub.

[0239] Clause 37. The method of clause 35, wherein the cells reside within the cannula for at least 5 minutes between loading and injection to allow cell sedimentation within the cannula hub.

[0240] Clause 38. The method of any of clauses 35-37, wherein the cannula is directed downward to allow cell sedimentation within the cannula hub.

[0241] Clause 39. The method of any of clauses 30 to 36, wherein the cell aggregates are photoreceptor progenitor cell aggregates.

[0242] Clause 40. The method of any of clauses 1 to 36, wherein the cells are retinal progenitor cells and / or photoreceptor progenitor cells.

[0243] Clause 41. The method of any of clauses 30 to 36, wherein the cell aggregates are retinal progenitor cells and / or photoreceptor progenitor cells.

[0244] Clause 42. The method of any of clauses 1 to 39, wherein the cells are injected in a volume of less than 200 μL.

[0245] Clause 43. The method of any of clauses 1 to 42, wherein the cells are injected in a volume of less than 100 μL.

[0246] Clause 44. The method of any of clauses 1-43, wherein the cells are injected in a volume of about 50 μL.

[0247] Clause 45. The method of any of clauses 1 to 44, wherein at least 1 million cells are injected.

[0248] Clause 46. The method of any of clauses 1 to 45, wherein at least 2 million cells are injected.

[0249] Clause 47. The method of any of clauses 1-46, wherein at least 25% of the cells loaded during the injection attachment are injected into the tissue of said subject.

[0250] Clause 48. The method of any of clauses 1-47, wherein at least 30% of the cells loaded during the injection attachment are injected into the tissue of said subject.

[0251] Clause 49. A method of treating an ocular condition in a subject, comprising cell transplantation in the eye of the subject, using an injection attachment comprising a cannula having a funnel-shaped hub.

[0252] Clause 50. The method of clause 49, wherein the cannula hub is less than 30 mm in length.

[0253] Clause 51. The method of clause 49 or 50, wherein the cannula includes a cannula tip of 30 gauge or less.

[0254] Clause 52. The method of clause 51, wherein the cannula tip is blunt.

[0255] Clause 53. The method of clause 51, wherein the cannula tip is sharp.

[0256] Clause 54. The method of clause 53, wherein the cannula tip is further defined as the needle tip.

[0257] Clause 55. The method of clause 51, wherein the cannula tip is a 34, 33, 32, 31 or 30 gauge cannula tip.

[0258] Clause 56. The method of clause 51, wherein the cannula tip is a 33 gauge cannula tip.

[0259] Clause 57. The method of any of clauses 51-56, wherein the cells are loaded into the cannula with a 30 gauge or smaller cannula tip.

[0260] Clause 58. The method of any of clauses 51-56, wherein the cells are loaded into the cannula by a 33 gauge cannula tip.

[0261] Clause 59. The method of any of clauses 51 to 56, wherein the cells are loaded into a cannula and injected into the eye of the subject through the tip of the same cannula.

[0262] Clause 60. The method of any of clauses 49 to 59, wherein the cannula tip is not altered between loading into the cannula and injecting into the subject's eye.

[0263] Clause 61. The method of any of clauses 49 to 60, wherein the cannula tip is made of a flexible polyimide material or metal.

[0264] Clause 62. The method of any one of clauses 49 to 61, wherein the cannula tip is flexible.

[0265] Clause 63. The method of any one of clauses 49 to 61, wherein the cannula tip is rigid.

[0266] Clause 64. The method of any of clauses 49 to 55, wherein the hub includes a one-way check valve.

[0267] Clause 65. The method of any of clauses 49 to 64, wherein the hub includes a coupling mechanism.

[0268] Clause 66. The method of clause 65, wherein the coupling mechanism is a luer lock.

[0269] Clause 67. The method of clause 66, wherein the luer lock is coupled to a dosing mechanism.

[0270] Clause 68. The method of clause 67, wherein the administration mechanism is a syringe.

[0271] Clause 69. The method of clause 68, wherein the syringe is a microinjection syringe.

[0272] Clause 70. The method of clause 68, wherein the syringe is further connected to a tubing system.

[0273] Clause 71. The method of clause 70, wherein the piping system is connected to a pressure control system.

[0274] Clause 72. The method of any of clauses 49 to 71, wherein the cells are delivered from the injection attachment at controlled pressure.

[0275] Clause 73. The method of any of clauses 49 to 72, wherein the cells are not redistributed prior to loading into the injection attachment.

[0276] Clause 74. The method of any of clauses 49 to 73, wherein the cells are redistributed prior to loading into the injection attachment.

[0277] Clause 75. The method of Clause 74, wherein the cells are redistributed by vortexing or manual agitation.

[0278] Clause 76. The method of any of clauses 49 to 76, wherein the cells are injected subretinal.

[0279] Clause 77. The method of any of clauses 49 to 76, wherein the cells are further defined as cell aggregates.

[0280] Clause 78. The method of any of clauses 49 to 76, wherein the cell is further defined as a single cell.

[0281] Clause 79. The method of any of clauses 49 to 78, wherein the cells are in a formulation buffer.

[0282] Clause 80. The method of Clause 79, wherein the formulation buffer is a balanced salt solution.

[0283] Clause 81. The method of Clause 80, wherein the balanced salt solution further comprises benzonase and / or human serum albumin.

[0284] Clause 82. The method of clause 77, wherein the cell aggregates are not in suspension when injected into the tissue of said subject.

[0285] Clause 83. The method of Clause 82, wherein the cells reside within the cannula for at least 5 minutes between loading and injection to allow aggregate sedimentation within the cannula hub.

[0286] Clause 84. The method of Clause 82, wherein the cells reside within the cannula for at least 5 minutes between loading and injection to allow cell sedimentation within the cannula hub.

[0287] Clause 85. The method of any of clauses 82-84, wherein the cannula is directed downward to allow cell sedimentation within the cannula hub.

[0288] Clause 86. The method of any of clauses 77 to 83, wherein the cell aggregates are photoreceptor progenitor cell aggregates.

[0289] Clause 87. The method of any of clauses 49 to 83, wherein the cells are retinal progenitor cells and / or photoreceptor progenitor cells.

[0290] Clause 88. The method of any of clauses 77 to 83, wherein the cell aggregates are retinal progenitor cells and / or photoreceptor progenitor cells.

[0291] Clause 89. The method of any of clauses 49 to 86, wherein the cells are injected in a volume of less than 200 μL.

[0292] Clause 90. The method of any of clauses 49 to 89, wherein the cells are injected in a volume of less than 100 μL.

[0293] Clause 91. The method of any of clauses 49-90, wherein the cells are injected in a volume of about 50 μL.

[0294] Clause 92. The method of any of clauses 49 to 91, wherein at least 1 million cells are injected.

[0295] Clause 93. The method of any of clauses 49 to 92, wherein at least 2 million cells are injected.

[0296] Clause 94. The method of any of clauses 49 to 93, wherein at least 25% of the cells loaded during the injection attachment are injected into the eye of said subject.

[0297] Clause 95. The method of any of clauses 49 to 94, wherein the ocular condition is damage caused by an inherited retinal disease, age-related macular degeneration (AMD), hereditary macular degeneration, Stargardt's macular dystrophy, Best's disease, choroideremia, diabetic retinopathy, retinal vascular disease, retinopathy of prematurity (ROP), or a viral infection of the eye.

[0298] Clause 96. A cannula device for cell transfer, comprising a tubular hub, the tubular hub comprising: The exterior wall and The interior walls and a first end; a second end and wherein the first end has a funnel shape tapering toward the second end.

[0299] Clause 97. The device of clause 96, wherein the inside diameter of the tubular hub decreases as measured from the first end toward the second end.

[0300] Clause 98. The device of clause 96, wherein the hub does not inherently have a catch point.

[0301] Clause 99. The device of clause 96, wherein the hub has a smooth surface.

[0302] Clause 100. Devices of clause 96 or 97, in which the tubular hub is less than 30 mm in length.

[0303] Clause 101. The device of any of clauses 96 to 100, wherein the first end is attached to the cannula tip.

[0304] Clause 102. The device of clause 101, wherein the cannula tip is blunt.

[0305] Clause 103. The device of clause 102, wherein the cannula tip is sharp.

[0306] Clause 104. The device of clause 103, wherein the cannula tip is further defined as a needle tip.

[0307] Clause 105. The device of clause 101, wherein the cannula tip is 30 gauge or less.

[0308] Clause 106. The device of clause 105, wherein the cannula tip is a 34, 33, 32, 31 or 30 gauge cannula tip.

[0309] Clause 107. The device of clause 105, wherein the cannula tip is a 33 gauge cannula tip.

[0310] Clause 108. The device of any of clauses 96 to 106, wherein the second end includes a one-way check valve integrated into the hub to prevent backflow.

[0311] Clause 109. The device of clause 108, wherein the valve is further connected to a piping system.

[0312] Clause 110. The device of any of clauses 101 to 109, wherein the cannula tip has an outer diameter ranging from about 0.30 mm to about 0.18 mm.

[0313] Clause 111. The device of any of clauses 101 to 110, wherein the cannula tip is made of a flexible polyimide material or metal.

[0314] Clause 112. The device of any of clauses 101 to 111, wherein the cannula tip is 1 mm, 2 mm, 3 mm, 4 mm or 5 mm in length.

[0315] Clause 113. The device of any one of clauses 101 to 112, wherein the cannula tip is flexible.

[0316] Clause 114. The device of any one of clauses 101 to 113, wherein the cannula tip is rigid.

[0317] Clause 115. The apparatus of any of clauses 101 to 114, wherein the cannula is attached to an infusion device.

[0318] Clause 116. The apparatus of any one of clauses 101 to 115, wherein the injection device is a syringe.

[0319] Clause 117. The device of clause 116, wherein the syringe includes a handle for adjusting the fluid flow path.

[0320] Clause 118. A device according to clause 116 or 117, wherein the syringe is a microinjection syringe.

[0321] Clause 119. A device according to any of clauses 116 to 118, wherein the syringe contains a volume of liquid of approximately 1 mL.

[0322] Clause 120. A device according to any one of clauses 101 to 119, wherein the tubular hub contains a liquid volume of less than 200 μL.

[0323] Clause 121. A device according to any of clauses 101 to 119, wherein the tubular hub contains a liquid volume of less than 100 μL.

[0324] Clause 122. The device of any of clauses 101 to 121, wherein the tubular hub contains a liquid volume of about 50 μL.

[0325] Clause 123. The device of any of clauses 101-122, wherein the cannula device is further defined as a subretinal delivery device.

[0326] Clause 124. A device according to any of clauses 101 to 123, used for the delivery of cells to a tissue of interest.

[0327] Clause 125. The device of any of clauses 101 to 123 for use in treating an ocular condition in a subject, comprising administering to the eye of said subject an effective amount of cells.

[0328] Clause 126. The use of clause 125, wherein the cells are loaded into a cannula and injected into the eye of the subject through the tip of the same cannula.

[0329] Clause 127. The use of clause 125, wherein the cannula tip is not altered between loading into the cannula and injecting into the subject's eye.

[0330] Clause 128. Use of clause 125, wherein the cells are delivered from the injection attachment at controlled pressure.

[0331] Clause 129. Use of any of clauses 126 to 128, wherein the cells are not redistributed before loading into the injection attachment.

[0332] Clause 130. Use of any of clauses 126 to 128, wherein the cells are redistributed before loading into the injection attachment.

[0333] Clause 131. The use of clause 130, wherein the cells are redistributed by vortexing or manual agitation.

[0334] Clause 132. The use of any of clauses 126 to 131, wherein the cells are injected subretinal.

[0335] Clause 133. The use of any of clauses 126 to 132, wherein the cell is further defined as a cell aggregate.

[0336] Clause 134. The use of any of clauses 126 to 133, wherein the cell is further defined as a single cell.

[0337] Clause 135. Use of any of clauses 126 to 134, wherein the cells are in a formulation buffer.

[0338] Article 136. Use of Article 135, wherein the formulation buffer is a balanced salt solution.

[0339] Clause 137. The use of Clause 136, wherein the balanced salt solution further comprises benzonase and / or human serum albumin.

[0340] Clause 138. The use of any of clauses 133 to 137, wherein the cell aggregates are not in suspension when injected into the tissue of said subject.

[0341] Clause 139. The use of any of clauses 133 to 138, wherein the cells are present in the cannula for at least 5 minutes between loading and injection to allow aggregate sedimentation within the cannula hub.

[0342] Clause 140. The use of any of clauses 133 to 138, wherein the cells are present in the cannula for at least 5 minutes between loading and injection to allow cell sedimentation within the cannula hub.

[0343] Clause 141. The use of any of clauses 133 to 140, wherein the cannula is directed downward to allow cell sedimentation within the cannula hub.

[0344] Clause 142. Use of any of clauses 133 to 139, wherein the cell aggregates are photoreceptor precursor cell aggregates.

[0345] Clause 143. Use of any of clauses 126 to 138, wherein the cells are retinal progenitor cells and / or photoreceptor progenitor cells.

[0346] Clause 144. Use of any of clauses 133 to 138, wherein the cell aggregates are retinal progenitor cells and / or photoreceptor progenitor cells.

[0347] Clause 145. Use of any of clauses 126 to 142, wherein the cells are injected in a volume of less than 200 μL.

[0348] Clause 146. Use of any of clauses 126 to 145, wherein the cells are injected in a volume of less than 100 μL.

[0349] Clause 147. The use of any of clauses 126 to 146, wherein the cells are injected in a volume of about 50 μL.

[0350] Article 148. Use of any of Articles 126 to 147, in which at least 1 million cells are injected.

[0351] Article 149. Use of any of Articles 126 to 148, in which at least 2 million cells are injected.

[0352] Clause 150. The use of any of clauses 126 to 149, wherein at least 25% of the cells loaded during the injection attachment are injected into the eye of said subject.

[0353] Article 151. The use of any of Articles 126 to 150, wherein the ocular condition is an inherited retinal disease, age-related macular degeneration (AMD), hereditary macular degeneration, Stargardt's macular dystrophy, Best's disease, choroideremia, diabetic retinopathy, retinal vascular disease, damage caused by retinopathy of prematurity (ROP), or a viral infection of the eye.

[0354] Clause 152. A kit comprising a device and cells according to any one of clauses 101 to 123.

[0355] Clause 153. A method of preparing a cell composition for delivery, comprising: (a) concentrating a cellular composition; (b) preparing a bulk dose of the cell composition; (c) dispensing a master dose from said bulk dose; (d) dispensing said dispensed doses; A method comprising:

[0356] Clause 154. The method of Clause 153, wherein the cell composition is a cryopreserved cell composition.

[0357] Clause 155. The method of Clause 153, further comprising thawing and resuspending the cryopreserved cell composition prior to step (a).

[0358] Clause 156. The method of any of clauses 153-155, further comprising dissociating and counting the cells in the cell composition after preparing the bulk dose.

[0359] Clause 157. The method of any of clauses 153 to 156, wherein step (c) comprises adding formulation buffer to reach a target concentration.

[0360] Clause 158. The method of any of clauses 153 to 157, wherein the bulk dose is 75 μL.

[0361] Clause 159. The method of any of clauses 153 to 158, wherein the dispensed volume is 50 μL.

[0362] Clause 160. The method of any of clauses 153-159, wherein the cell composition is resuspended in a formulation buffer.

[0363] Clause 161. The method of any of Clauses 160, wherein the formulation buffer comprises a balanced salt solution.

[0364] Clause 162. The method of clause 160 or 161, wherein the formulation buffer further comprises albumin.

[0365] Clause 163. The method of Clause 162, wherein the albumin is human serum albumin.

[0366] Clause 164. The method of any of clauses 153-163, further comprising loading a cell composition into the device of any of clauses 96-123.

[0367] Clause 165. The method of any of clauses 153-163, further comprising delivering a cell composition according to the method of any of clauses 1-95.

[0368] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. U.S. Patent Application Publication No. 2002 / 0076747 International Publication WO98 / 30679 Remington's The Science and Practice of Pharmacy,21 st Edition,AR Gennaro(Lippincott,Williams&Wilkins,Baltimore,MD,2006 Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Lab. Press, 2001.

Claims

1. 1. A method of cell transplantation using an injection attachment having a hub containing a plurality of cells and a cannula extending from a distal end of the hub, comprising: orienting the injection attachment so that the cannula points downward until the cells settle together adjacent the distal end; inserting the cannula into tissue of a subject; injecting the plurality of cells into the tissue of the subject using the injection attachment; A method comprising:

2. The method of claim 1 , wherein the hub includes a funnel-shaped inner surface extending between the distal and proximal ends.

3. 3. The method of claim 2, wherein the inner surface tapers inwardly from the proximal end to the distal end such that the diameter of the inner surface is smaller at the distal end than the diameter of the inner surface at the proximal end.

4. The method of claim 2 , wherein the inner surface of the hub does not overlap the cannula.

5. 10. The method of claim 1, wherein the injection attachment is oriented so that the cannula points downward for at least 30 seconds.

6. 10. The method of claim 1, wherein the injection attachment is oriented so that the cannula points downward for at least two minutes.

7. 10. The method of claim 1, wherein the injection attachment is oriented so that the cannula points downward for at least five minutes.

8. The method of claim 1 , wherein at least 99% of the plurality of cells in the hub are infused into the tissue.

9. 10. The method of claim 1, wherein the injection attachment is held at an angle of greater than 45 degrees relative to the ground while injecting the plurality of cells into the tissue of the subject.

10. The method of claim 1 , further comprising providing a plurality of cells to the injection attachment so that the hub is pre-loaded.

11. 11. The method of claim 10, wherein the plurality of cells are drawn into the hub through the distal end of the cannula prior to injection and injected through the cannula.

12. 12. The method of claim 11, wherein the cannula is not altered between providing the plurality of cells into the hub and injecting the cells into the tissue of the subject.

13. The method of claim 1 , wherein the cannula includes a cannula shaft and a cannula tip extending from a distal end of the cannula shaft.

14. The method of claim 13, wherein the cannula tip is a 33 gauge cannula tip.

15. The method of claim 1 , wherein the cannula tip is blunt.

16. The method of claim 1 , wherein the tissue is retinal tissue.

17. The method of claim 1 , wherein the cells are stem cells or stem cell-derived cells.

18. 1. A method of cell transplantation in an eye using an injection attachment having a hub containing a plurality of cells and a cannula extending from a distal end of the hub, comprising: orienting the injection attachment so that the cannula points downward until the cells settle together adjacent the distal end; inserting the cannula into retinal tissue of a subject; injecting the plurality of cells into the retinal tissue of the subject using the injection attachment; Including, the injection attachment is oriented so that the cannula points downward for at least two minutes; the hub includes an engagement end and a delivery end and an interior surface extending between the engagement end and the delivery end; The method wherein the inner surface tapers inwardly between the engagement end and the delivery end.

19. 20. The method of claim 18, wherein the plurality of cells is provided to the hub and injected through the cannula.

20. 1. An injection attachment for cell transplantation in the eye, comprising: a hub having a proximal end, a distal end, and an interior surface extending between the proximal and distal ends and defining an interior volume; a cannula coupled to the distal end of the hub in fluid communication with the interior volume; Including, the interior volume contains a pre-loaded amount of cells; the interior volume is generally funnel-shaped; the inner surface of the hub does not overlap the cannula; An injection attachment wherein when the distal end of the hub faces downward, at least 90% of the cells settle proximate to the distal end of the hub.

21. The hub includes: a body defining the proximal end of the hub; a bushing coupled to an inner surface of the body and defining the distal end of the hub; 21. The injection attachment of claim 20, comprising:

22. the inner surface tapers inwardly from the proximal end to the distal end such that the diameter of the inner surface is smaller at the distal end than the diameter of the inner surface at the proximal end; 21. The injection attachment of claim 20, wherein the interior volume does not retain cells after injection.

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