Devices and methods for delivery of cellular compositions

Abstract

Provided herein are methods of cell transplantation using an injection attachment having a hub comprising a plurality of cells and a cannula extending from a distal end of the hub. The method includes orienting the injection attachment such that the cannula points downward until the plurality of cells settle together near the distal end, inserting the cannula into the tissue of the subject, and injecting a volume of cells into the tissue of the subject using the injection attachment.

Description

[0001] Priority Claim

[0002] This application claims the benefit of priority of U.S. Provisional Application Serial No. 63 / 412,195, filed Sep. 30, 2022, the entire content of which is hereby incorporated by reference. Field of the Invention

[0003] The present invention generally relates to the field of cell therapy. More specifically, it relates to devices and methods for delivering cell compositions to a subject and methods of using the same. Background of the Invention

[0004] Cell therapy involves the transfer of autologous or allogeneic cell material into a patient for medical purposes. Cell-based therapies involve delivering cells into the circulation or tissue to treat a variety of diseases. Today, cell therapy continues to evolve with ongoing research into clinical safety and efficacy. Cell therapy combines single-cell or multi-cell therapies based on stem cells and non-stem cells. It typically uses autologous or allogeneic cells; may involve genetic engineering or manipulation in formulations; and can be administered locally or as an injection, infusion, biologic scaffold, or scaffold-free system. Cell therapy spans multiple therapeutic areas such as regenerative medicine, immunotherapy, and cancer therapy. Currently, most cell therapies are in the early stages of development (phase 1 / 2), with a few exceptions being current best practices in specific situations (e.g., bone marrow / stem cell transplantation, hepatocyte transplantation, skin equivalents), or being approved for specific indications such as (sipuleucel-T), (azficel-T), (autologous cultured chondrocytes on porcine collagen) and KYMRIAH TM (tisagenlecleucel) etc. (El-Kadiry et al., Front. Med., 8; 2021).

[0005] Pluripotent stem cells (PSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have been used to study the development of disease processes and as potential therapies for multiple organ systems. In recent years, there has been increasing interest in using PSC-based transplantation to treat retinal diseases in which retinal ganglion cells have become functionally impaired or lost due to degeneration. Preclinical trials in animal models of retinal diseases have shown improvement in visual outcomes after subretinal transplantation of PSC-derived photoreceptors or retinal pigment epithelium (RPE) cells. Death of retinal photoreceptor cells (PRs; rods and cones) due to inherited retinal diseases (IRDs) or injury is a major cause of untreatable blindness worldwide. IRDs (also known as photoreceptor degenerative diseases) can present as primarily rod PR loss, primarily cone PR loss, or 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 (iPRPs) as a cell-based PR replacement therapy for IRDs.

[0006] For cell-based therapies, successful translation of preclinical research into clinical practice requires effective means of cell delivery on a human patient scale. There are many injection strategies for cell-based therapies. Direct injection involves using a needle and syringe or injecting cells into the body through a delivery device such as a port or catheter / reservoir system. Intravenous injection is the most accessible injection route for patients and involves infusion into the bloodstream. Transplanted cells are typically delivered with a guiding straight cannula or needle. Usually, the cannula is coupled to some form of syringe for dispensing the cell suspension.

[0007] Problems with the delivery of cell therapies include the unintended deposition of cells to non-target sites, unpredictable cell doses at the intended target site, and even substantial losses of the cell suspension. The cell delivery cannula is typically connected to an external syringe via a Luerlock or similar coupling mechanism. This design has several drawbacks. First, for most syringes, a small movement of the plunger dispenses a relatively large volume, making it difficult to manually achieve the delivery of a small, precise dose of cell suspension. To address this issue, it has been shown beneficial to incorporate mechanical or electrical drive to control the translational movement of the plunger. Second, the mechanical forces at the transition point between the syringe and the catheter can damage the cells. The inner diameter of the syringe is typically larger than that of the injection cannula. Thus, when cells and fluid pass from the syringe to the cannula, their linear velocity experiences a significant increase. This creates a differential velocity along the cell length, called the tensile force, which is thought to be an important cause of cell damage during injection. Additionally, the cells are also exposed to shear stress as the cells and fluid in the middle of the cannula move at a higher velocity than at the outer boundary (Potts et al., Surg Neurol Inst., 4(S22 - S30):2013).

[0008] Many cell therapy methods aim to deliver a high-density single cell suspension to diseased or injured sites in the body. However, even in non-adhesive matrices, cell aggregates maintain viability, cell activity, and phenotype beyond that of individual cells, enabling the delivery of higher cell densities with enhanced proliferative and differentiative capabilities. Thus, there is an unmet need for improved methods and devices for delivering cell therapies, particularly cell aggregates, without damaging or losing cells and delivering a precise high-density dose to the target tissue. Summary of the Invention

[0010] In a first embodiment, the present invention provides a method of cell transplantation, which includes injecting cells into a tissue of a subject using a cell delivery device, the cell delivery device including a cannula and a funnel shaped hub.

[0011] In certain aspects, the hub of the present invention is substantially free of capture points that would increase shear or turbulence as cells move through the hub. In certain aspects, the hub is substantially smooth as cells pass through the hub and enter the cannula shaft. In some aspects, the reduction of turbulence or shear helps to improve cell viability, while capture points can result in cell waste and damage. In a specific aspect, compared to a hub that does not have a funnel shape, the hub of the present invention is substantially free of capture points and is substantially smooth to allow for the expulsion of increased cell material (i.e., less material remains in the hub).

[0012] In some aspects, the length of the cannula hub is less than 30 mm. In some aspects, the cannula includes a cannula tip of 30 gauge or smaller. 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-gauge, 33-gauge, 32-gauge, 31-gauge, or 30-gauge cannula tip. In some aspects, the cannula tip is a 33-gauge cannula tip. In certain aspects, cells are loaded into the cannula with a cannula tip of 30 gauge or smaller. In some aspects, cells are loaded into the cannula with a 33-gauge cannula tip. In some aspects, cells are loaded into the cannula and injected into the tissue of the subject with the same cannula tip. In certain aspects, the cannula tip is not replaced between loading the cells into the cannula and injecting the cells into the tissue of the subject (e.g., the eye).

[0013] In some aspects, the cannula tip is made of a flexible polyimide material or metal. In some aspects, the cannula tip is flexible. In certain aspects, the cannula tip is rigid.

[0014] 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 connected to a drug delivery mechanism. A controlled pressure can be applied to the drug delivery mechanism to deliver a dose. In some aspects, the drug delivery mechanism is a syringe. In some aspects, the syringe is a microinjection syringe. In certain aspects, the syringe is further connected to a conduit. In some aspects, the conduit is connected to a pressure control system. In certain aspects, cells are delivered from a cell delivery device under a controlled pressure. In some aspects, the cells are not redistributed or resuspended before being loaded into the cell delivery device. In some aspects, the cells are redistributed or resuspended before being loaded into the cell delivery device. In certain aspects, the cells are redistributed or resuspended by vortexing or manual agitation.

[0015] In some aspects, cells are injected into the eye of the subject. In certain aspects, the cells are injected subretinally. 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 suspended when injected into the tissue of the subject. In certain aspects, the cells are present in 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, such as 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 the aggregates to settle in the cannula hub. In certain aspects, the cells are present in 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, such as 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 the cells (such as single cells) to settle in the cannula hub. In certain aspects, the cannula is pointed downward to allow the cells to settle in the cannula hub.

[0016] In some aspects, the cell aggregates are photoreceptor progenitor cell aggregates. In some aspects, the cells are retinal pigment epithelium (RPE) and / or photoreceptor progenitor cells. In some aspects, the cell aggregates are RPE and / or photoreceptor progenitor cells. In some aspects, the cells are injected in a volume less than 200 uL. In some aspects, the cells are injected in a volume less than 100 uL. In some aspects, the cells are injected in a volume of about 50 uL (e.g., about 25 - 50 uL, 50 - 75 uL, 75 - 100 uL, 100 - 150 uL, or 150 - 200 uL, such as about 25, 50, 75, 100, 125, 150, 175, or 200 uL). In some aspects, at least 100,000, 250,000, 500,000, or 1 million cells are injected. In certain aspects, 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 aspects, at least 25% of the cells loaded into the cell delivery device are injected into the tissue of the subject. In some aspects, 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 tissue of the subject.

[0017] Another embodiment provides a method for treating an eye disorder in a subject, which includes performing cell transplantation in the subject's eye using a cell delivery device, the cell delivery device including a cannula and a funnel-shaped hub. In some aspects, the cells are injected subretinally. In some aspects, the length of the cannula hub is less than 30 mm. In certain aspects, the cannula includes a cannula tip of 30 gauge or smaller. 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-gauge, 33-gauge, 32-gauge, 31-gauge, or 30-gauge cannula tip. In a particular aspect, the cannula tip is a 33-gauge cannula tip. In some aspects, the cells are loaded into the cannula using a cannula tip of 30 gauge or smaller. In certain aspects, the cells are loaded into the cannula using a 33-gauge cannula tip.

[0018] In some aspects, the cells are loaded into the cannula and injected into the subject's eye using the same cannula tip. In some aspects, the cannula tip is not replaced between loading the cannula and injecting into the subject's eye. In some aspects, the cannula tip is made of a flexible polyimide material or metal. In some aspects, the cannula tip is flexible. In certain aspects, the cannula tip is rigid.

[0019] In some 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 connected to a drug delivery mechanism. In some aspects, the drug delivery mechanism is a syringe. In some aspects, the syringe is a microinjection syringe. In certain aspects, the syringe is also connected to a conduit. In some aspects, the conduit is connected to a pressure control system.

[0020] In certain aspects, cells are delivered from a cell delivery device under controlled pressure. In some aspects, the cells are not redistributed or resuspended prior to loading into the cell delivery device. In certain aspects, the cells are redistributed or resuspended prior to 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 subretinally injected.

[0021] 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 specific aspects, the cell aggregates are not suspended when injected into the tissue of the subject. In some aspects, the cells are present in 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, such as 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 the cells (e.g., aggregates) to settle in the cannula hub. In specific aspects, the cannula is pointed downward to allow the cells to settle in the cannula hub. In some aspects, the cell aggregates are photoreceptor progenitor cell aggregates. In certain aspects, the cells are retinal progenitor cells (RPE) and / or photoreceptor progenitor cells. In some aspects, the cell aggregates are RPE and / or photoreceptor progenitor cells. In certain aspects, the cells are injected in a volume of less than 200 uL. In some aspects, the cells are injected in a volume of less than 100 uL. In some aspects, the cells are injected in a volume of about 50 uL (e.g., about 25 - 50 uL, 50 - 75 uL, 75 - 100 uL, 100 - 150 uL, or 150 - 200 uL, such as about 25, 50, 75, 100, 125, 150, 175, or 200 uL). In specific aspects, at least 100,000, 250,000, 500,000, or 1 million cells are injected. In certain aspects, 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 aspects, at least 25% of the cells loaded into the cell delivery device are injected into the eye of the subject. In some aspects, the eye disorder is an injury, a hereditary retinal disease, age - related macular degeneration (AMD), hereditary macular degeneration, Stargardt macular dystrophy, Best's disease, choroideremia, diabetic retinopathy, retinal vascular disease, an injury caused by retinopathy of prematurity (ROP), or a viral infection of the eye. The eye injury can include an injury caused by a scratch, a stab wound, a scrape, a penetrating wound, a perforating wound, or an intraocular foreign body.

[0022] Another embodiment provides a cannula device for cell transplantation, which includes a tubular hub, wherein the tubular hub includes: an outer wall; an inner wall; a first end; and a second end, wherein the first end has a funnel shape that slopes towards the second end. In some aspects, the inner diameter of the tubular hub gradually decreases when measured from the first end towards the second end. In certain aspects, the hub is substantially free of capture points. In certain aspects, the hub has a smooth surface.

[0023] In certain aspects, the hub of the present invention is substantially free of capture points that would increase shear or turbulence when cells move through the hub. In certain aspects, the hub is substantially smooth when cells pass through the hub and enter the cannula shaft. In particular aspects, the reduction of turbulence or shear helps to improve cell viability, while capture points can lead to cell waste and damage. In specific aspects, compared to a non-funnel-shaped hub, the hub of the present invention is substantially free of capture points and is substantially smooth to allow for the expulsion of increased cell material (i.e., less material remains in the hub).

[0024] In some aspects, the first end is attached to the cannula tip. In certain aspects, the cannula tip is blunt. In some aspects, the cannula tip is sharp. In some aspects, the cannula tip is further defined as a needle tip.

[0025] In some aspects, the length of the tubular hub is less than 30 mm. The cannula tip is 30 gauge or smaller. In certain aspects, the cannula tip is a 34-gauge, 33-gauge, 32-gauge, 31-gauge, or 30-gauge cannula tip. In some aspects, the cannula tip is a 33-gauge cannula tip. In some aspects, the second end includes a one-way check valve integrated in the hub that prevents backflow.

[0026] In particular aspects, the valve is further connected to a conduit. In some aspects, the cannula tip has an outer diameter range of from about 0.30 mm to about 0.18 mm. In some aspects, the cannula tip is made of a flexible polyimide material or metal. In certain aspects, the length of the cannula tip is 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm.

[0027] In some aspects, the cannula tip is flexible. In certain aspects, the cannula tip is rigid. In some aspects, the cannula is attached to an injection device. In certain aspects, the injection device is a syringe. In certain aspects, the syringe includes a handle for regulating the fluid flow path. In specific aspects, the syringe is a microinjection syringe. In some aspects, the syringe includes a fluid volume of about 1 mL. In some aspects, the tubular hub includes a fluid volume of less than 200 uL. In specific aspects, the tubular hub includes a fluid volume of less than 100 uL. In some aspects, the tubular hub includes a fluid volume of about 50 uL (e.g., about 25 - 50 uL, 50 - 75 uL, 75 - 100 uL, 100 - 150 uL, or 150 - 200 uL, such as about 25, 50, 75, 100, 125, 150, 175, or 200 uL). In some aspects, the cannula device is further defined as a subretinal delivery device.

[0028] Other embodiments provide for the use of the devices of the present invention in delivering cells to a subject's tissue. Also provided herein is the use of the devices of the present invention in treating an ocular disorder of a subject, including administering to the subject's eye an effective amount of cells. In some aspects, cells are loaded into the cannula and injected into the subject's eye with the same cannula tip. In certain aspects, the cannula tip is not replaced between loading into the cannula and injection into the subject's eye. In some aspects, cells are delivered from the cell delivery device under controlled pressure. In certain aspects, the cells are not redistributed or resuspended prior to loading into the cell delivery device. In some aspects, the cells are redistributed or resuspended prior to loading into the cell delivery device. In some aspects, the cells are redistributed or resuspended by vortexing or manual agitation. In certain aspects, the cells are injected subretinally. In some aspects, a pre-bleb is created with a different cannula prior to injecting the cells with the funnel-shaped hub. 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 specific aspects, the formulation buffer is a balanced salt solution. In particular aspects, the balanced salt solution further contains benzonase and / or human serum albumin.

[0029] In some aspects, when injected into the tissue of the subject, the cell aggregates are not suspended. In some aspects, between loading and injection, the cells are present in the cannula for at least 5 minutes to allow the aggregates to settle in the cannula hub. In some aspects, between loading and injection, the cells are present in 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, such as 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) to allow the cells (such as single cells) to settle in the cannula hub. In certain aspects, the cannula is pointed downward to allow the cells to settle in the cannula hub. In some aspects, the cell aggregates are photoreceptor progenitor cell aggregates. In some aspects, the cells are retinal progenitor cells (RPE) and / or photoreceptor progenitor cells. In some aspects, the cell aggregates are RPE and / or photoreceptor progenitor cells. In some aspects, the cells are injected in a volume less than 200 uL. In some aspects, the cells are injected in a volume less than 100 uL. In certain aspects, the cells are injected in a volume of about 50 uL (e.g., about 25-50 uL, 50-75 uL, 75-100 uL, 100-150 uL, or 150-200 uL, such as about 25, 50, 75, 100, 125, 150, 175, or 200 uL). In some aspects, at least 100,000, 250,000, 500,000, or 1 million cells are injected. In certain aspects, 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 aspects, at least 25% of the cells loaded into the cell delivery device are injected into the eye of the subject. In some aspects, the ocular disorder is a hereditary retinal disease, age-related macular degeneration (AMD), hereditary macular degeneration, Stargardt macular dystrophy, Best disease, choroideremia, diabetic retinopathy, retinal vascular disease, damage caused by retinopathy of prematurity (ROP), or a viral infection of the eye.

[0030] The present disclosure also provides kits that include the devices and cells of the embodiments and aspects of the present invention.

[0031] The present invention also provides a method for cell transplantation using an injection attachment, the 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 such that the cannula points downward until the plurality of cells settle together near the distal end; inserting the cannula into a tissue of a subject; and injecting the plurality of cells into the tissue of the subject using the injection attachment.

[0032] In certain embodiments, the hub includes a funnel-shaped inner surface extending between a distal end and a 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 at the distal end is less than the diameter of the inner surface at the proximal end. In certain embodiments, the inner surface of the hub does not overlap with the cannula.

[0033] In certain embodiments, at least 90% of the cells settle together near the distal end. In certain embodiments, the injection attachment is oriented such that the cannula points downward for at least 2 minutes. In certain embodiments, when injecting the plurality of cells into the tissue of the subject, the injection attachment is held at an angle greater than 45 degrees.

[0034] The method may further comprise: providing a plurality of cells to the injection attachment such that the hub is pre-filled. In certain embodiments, prior to injection, the plurality of cells are aspirated into the hub through the distal end of the cannula and injected through the cannula. In certain embodiments, the cannula is not replaced between providing the plurality of cells to the hub and injecting the cells into the tissue of the subject.

[0035] In certain embodiments, the cannula includes a cannula shaft and a cannula tip extending from its distal end. In certain embodiments, the cannula tip is a 33-gauge cannula tip. In certain embodiments, the cannula tip is blunt-ended. In certain embodiments, the tissue is retinal tissue. In certain embodiments, the cells are stem cells.

[0036] The present invention also provides a method for cell transplantation in an eye using an injection attachment, the 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 such that the cannula points downward until the plurality of cells settle together near the distal end; inserting the cannula into the retinal tissue of a subject; and injecting the plurality of cells into the retinal tissue of the subject using the injection attachment. In certain embodiments, the injection attachment is oriented such that the cannula points downward for at least 2 minutes. In certain embodiments, the hub includes a mating end and a delivery end and an inner surface extending between the mating end and the delivery end. In certain embodiments, the inner surface tapers inwardly between the mating end and the delivery end. In certain embodiments, a plurality of cells are provided to the hub and injected through the cannula.

[0037] The present disclosure also provides an injection accessory for cell transplantation in an eye, comprising: a hub having a proximal end, a distal end, and an inner surface extending therebetween, the inner surface defining an internal volume; and a cannula coupled to the distal end of the hub and in fluid communication with the internal volume. In some embodiments, the internal volume comprises a pre-filled volume of stem cells. In some embodiments, the internal volume is generally funnel-shaped. In some embodiments, at least 90% of the cells settle near the distal end of the hub when the distal end of the hub points downward. In some embodiments, the hub comprises: 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 some embodiments, the inner surface tapers inwardly from the proximal end to the distal end such that the diameter of the inner surface at the distal end is less than the diameter of the inner surface at the proximal end. In some embodiments, the internal volume does not retain stem cells after injection.

[0038] Other objects, features, and advantageous aspects of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the 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. Brief Description of the Drawings

[0040] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings in conjunction with the detailed description of specific embodiments given herein.

[0041] Figure 1 : Side view of the injection accessory 100.

[0042] Figure 2 : Partial cross-sectional view of the injection accessory 100.

[0043] Figure 3 : Flowchart of rodent and clinical / NHP formulations.

[0044] Figure 4 : The dosing mechanism 200 (e.g., Vision System) is attached to the tubing from the viscous fluid control package 210, MICRODOSE TM Injection Kit 220, and the injection accessory 100. Sometimes, a manual syringe (i.e., a manually operated plunger) and different cannula tips and cannulas can be used.

[0045] Figures 5A - 5C : For the injection accessory 100, the specifications do not affect the integrity of the cell aggregates. ( Figure 5A) Two users loaded iPRP cell aggregates and expelled them through a 31G or 33G cannula to determine if there was any detrimental effect on the aggregate product when passing through a smaller cannula. Neither the volume % nor the mean diameter of the gate was significantly affected by the cannula size. The volume percentage of biomass over 17.03 diameter was calculated by obtaining the biomass over 17.03 diameter and dividing it by the total biomass (left / primary y-axis). ( Figure 5B ) Comparison of the median diameter of iPRP aggregates after no injection, after using the dosing mechanism 200 (e.g., Constellation) to inject through the injection attachment 100 to increase the pressure to 10 PSI, hold, then increase to 16 PSI and release, or after injection at 16 PSI. ( Figure 5C ) Comparison of the aggregate biomass percentage of iPRP aggregates after no injection, after using the dosing mechanism 200 (e.g., Constellation) to inject through a custom cannula to increase the pressure to 10 PSI, hold, then increase to 16 PSI and release, or after injection at 16 PSI.

[0046] Figure 6 : The iPRP aggregates can be kept in suspension to ensure consistent dose delivery. Injections 1 - 34 show the percentage of expected cell recovery according to the standard FCDI test bench test dose injection protocol. The low recovery dose at the surgeon's orientation (injection #35) may be attributed to the settling of the dose in the syringe due to the time and angle the syringe was held. Test #36 (5 - minute rolling dose) indicates that the low recovery rate at the surgeon's orientation is not solely attributed to the time the dose was held in the syringe. The expected cell percentages are shown. For example, if 1M cells were recovered but the 1M dose was the expected target, it would be 100% as expected, but if the 2M dose was the expected target and only 1M cells were recovered, the recovery would be 50% as expected.

[0047] Figure 7 : Flicking the syringe loaded with photosensitive cell aggregates can redistribute the settled aggregates and deliver an appropriate dose with a standard cannula. When using a standard cannula, the cell aggregates may fall out of the formulation, so attempts were made to determine ways to maintain proper aggregate distribution in the syringe. Flicking, vortexing, or not mixing with a standard cannula such as Figure 7As shown. After the dose is generated, a standard syringe is prepared to inject 2 million cells per 50 μL (NHP dose). The syringe is then held in a horizontal position for 5 minutes, after which the dose is mixed or not mixed according to the test conditions. It can be seen that there is a significant lack of recovered cells under non-mixing conditions, while approximately 2 million target cells are recovered under vortexing and flicking conditions. Therefore, without effective mixing with the standard cannula, sufficient dosing cannot be achieved, and variability in surgical injection remains.

[0048] Figures 8A - 8F : Injection with the injection attachment 100 and simulated injection with aggregation sedimentation deliver a more consistent dose than the suspension method. ( Figure 8A ) Allowing the iPRP aggregates to sediment in an off-the-shelf cannula (MedOne #3262) impedes the correct delivery of the dose. ( Figure 8B ) Technical drawing of an off-the-shelf 33G cannula (MedOne #3262). ( Figure 8C ) Image of the aggregates (white ring) trapped in the off-the-shelf cannula hub after fully depressing the plunger. The arrows in B and C show the cannula hub 301 in which the aggregates are captured. ( Figure 8D ) Technical drawing of the funnel-shaped hub 110 of the injection attachment 100. ( Figure 8E and Figure 8F ) Two experiments comparing the injection attachment 100 and an off-the-shelf cannula showed that the sedimentation method delivers a more consistent dose than the suspension method.

[0049] Figure 9 : Passing the iPRP aggregates through a 33G cannula twice does not significantly affect the aggregate size compared to a single pass. After loading the dose into the syringe through an 18G needle (single pass) or through a 33G cannula (two passes), two users expelled the photoreceptor cell aggregates through a standard 33G cannula. These aggregates were then analyzed on a Multisizer to determine if there was any effect on aggregate integrity. Neither the average diameter nor the volume % in the gate was significantly affected by the number of times the aggregates passed through the cannula.

[0050] Figure 10 : Schematic diagram of dose preparation for concentration studies. After thawing and washing the iPRP aggregated cells, they are resuspended at a concentration higher than the target concentration. Samples are taken from this bulk for cell counting. Once the concentration of this bulk is determined, it is aliquoted into separate tubes and sufficient medium is added to dilute the master dose to its final concentration. These master doses are then sub-aliquoted into equal portions intended for injection.

[0051] Figure 11: Master dose variability from concentration studies. For the injection attachment 100, in 10 experiments, the average expected recovery percentage of 28 different iPRP aggregate master doses was 93%, with a coefficient of variation of 11.4%.

[0052] Figure 12 : Schematic of final dose preparation. After thawing and washing the iPRP aggregated cells, they are resuspended at a concentration higher than the target concentration. Samples are taken for cell counting, the bulk volume (except for 20 μL) is transferred to a separate tube, and sufficient medium is added to dilute the master dose to the final concentration. The master dose is then aliquoted for injection.

[0053] Figure 13 : Master dose variability generated by the clinical workflow. The master dose (1 donor to 1 master dose) of iPRP aggregates prepared using the clinical workflow was on average 93.5% of the expected cell concentration, with a coefficient of variation of 5.0%.

[0054] Figure 14 : The coefficient of variation between doses of the administered dose was 7.5% to 15.5%. In 8 experiments using the injection attachment 100 in which the master dose was generated from a single donor preparation of iPRP aggregates, 5 and 12 aliquots with a volume range of 60 to 210 μL were taken and dissociated for counting to calculate the percentage of expected recovery and determine the variability between doses. All doses were within ±55% of their expected cell concentration.

[0055] Figures 15A - 15C : Dose recovery at different cell concentrations. After observing that the cell recovery rate from injection was lower than expected, the calculated doses were queried within a certain concentration range using the injection attachment 100. ( Figure 15A ) After preparing 82 calculated doses in 11 experiments and 6 batches, it was determined that to inject 1 million cells (clinical dose), a dose of 1.7 million cells was required, and to inject 2 million cells (NHP dose), a dose of 2.8 million cells was required. ( Figure 15B ) For the dose of 1.7 million cells, in 6 experiments, in 3 batches and 25 injections, on average, the injection delivered 1.04 million cells, with a coefficient of variation of 19%. 84% (21 / 25) of the doses fell within ±30% of the target of 1 million cells. ( Figure 15C) For a dose of 2.8 million cells, in 3 experiments, across 3 batches and 15 injections, the average delivered injection contained 2.1 million cells, with a coefficient of variation of 16%. 93% (14 / 15) of the doses fell within ±30% of the target of 2 million cells. For doses of 3 million and 4 million cells, a linear fit line was extrapolated from all data points generated from 391 individual injections, which spanned 2 iPSC cell lines, 8 iPRP batches (16 sub-batches) for all 4 doses, and the best fit equation was y = 0.0348x - 0.1239. Using this equation, doses of 4.5 million and 5.95 million cells were calculated to be required to load to achieve target doses of 3 million and 4 million cells injected, respectively.

[0056] Figure 16A - Figure 16C : Immunofluorescence images demonstrated the survival and photoreceptor identity of transplanted cells injected into the eye above the fovea of the retina. ( Figure 16A - Figure 16C ) Representative micrographs of the transplanted cell area (multiple areas imaged). Stem121 labels the cytoplasm of all transplanted human cells (note that the host RPE is also highly autofluorescent). AIPL1 labels rod and cone photoreceptor cells. AIPL1 has some cross-reactivity with host NHP photoreceptors, however, the labeling of transplanted human photoreceptors is much brighter. In a normal retina with an intact ONL and outer limiting membrane, iPRP cell aggregates are mainly located in the subretinal space and do not integrate into the host ONL as expected. A'-C') Corresponding brightfield (DIC images). Transplanted cells often contact the pigmented RPE (the brown cells surrounding the AIPL1 / Stem121 staining). It is unclear whether this is an artifact of thick frozen sections or a biological response to xenotransplantation. Note that the separation of the neurosensory retina is an artifact of tissue processing and sectioning. T = transplanted cell area. ONL = outer nuclear layer. INL = inner nuclear layer.

[0057] Figure 17A : Figure 1 Side view cross-sectional view of injection attachment 100 coupled to a syringe, the injection attachment containing a volume of cells in the distal end of the funnel-shaped hub 110.

[0058] Figure 17B : Figure 1 Side view cross-sectional view of injection attachment 100 coupled to a syringe, the injection attachment containing a volume of cells in the distal end of the funnel-shaped hub 110 and inserted into the eye of a subject. DETAILED DESCRIPTION OF THE INVENTION

[0060] I. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0061] 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 damaging or losing cells. The integrity of cell compositions can be negatively impacted by the way they are handled (such as processing steps involving multiple needle changes). Cell compositions can include single cell suspensions or cell aggregates and can be single-celled or multi-cellular. The cells in the composition can settle out of the formulation buffer, which can affect issues during dose formulation and dose delivery. In particular, standard cannulas cannot be used to reliably provide precise dose delivery because cells in the composition can be trapped in the cannula hub due to settling and inappropriate resuspension techniques. Therefore, it is hypothesized that a custom cannula with a hub shaped like a funnel will improve injection dose consistency because there will be substantially no space for trapping cell components (such as single cells or aggregates).

[0062] In some aspects, cell aggregates can have a median aggregate diameter of about 40 - 60 μm, such as 40 - 45 μm, 45 - 55 μm, 50 - 55 μm, or 50 - 60 μm. The integrity of the aggregates can be determined by measuring aggregate diameter and volume % > 17.03um. In some aspects, the aggregate size can be between 10 μm and 200 μm, such as between 15 μm and 170 μm or 17 μm and 168 μm.

[0063] Known cell compositions, such as cell aggregates, can fall out of the formulation buffer once loaded into a syringe, resulting in inaccurate injection doses, and thus methods for maintaining proper aggregate distribution in the syringe were sought. To prevent aggregate settling, flicking and vortex testing were tested and shown to be ([ Figure 7 ) effective for mixing with standard cannulas. In this "resuspension" method, an excess volume is drawn into a syringe attached to the cannula, air bubbles are removed by flicking and ejecting the unwanted volume, and then the remaining volume is injected. The problem with this method is that cells (such as aggregates) can settle because they are not evenly distributed throughout the volume. Thus, the resuspension method results in non-uniform cell suspension, which leads to over-dosing or under-dosing. Additionally, even with flicking or vortexing, loading and injection need to be done quickly, and even then, variability in the cell composition delivered during a surgical injection still exists.

[0064] For this reason, it was investigated whether purposefully allowing aggregates to settle would permit more consistent dosing. However, studies of standard cannulas showed that syringes that allowed cells to settle in them injected less than 25% of the expected dose. It was found that standard syringes ([ Figure 8B ) include a cannula hub 301 that can trap cells and prevent them from being injected, resulting in a lower dose being administered.

[0065] Since the aggregates are allowed to settle towards the tip of the cannula, which was initially inoperative due to the geometry of the cannula hub, the injection attachment 100 (as referred to herein, and as referred to as the cannula or custom cannula design in the applications claiming priority to the present disclosure), using a new hub that functions more like a funnel and a cannula extending distally from the hub, provides a more consistent dose. The cannula of the present invention may include a continuous funnel-shaped flow pattern that reduces drag and turbulence as the liquid passes through the cannula. With this newly designed cannula, a lower dose (e.g., 50 ul) can be loaded through the cannula, allowed to settle towards its tip for 5 minutes, and then injected, eliminating the need to keep the aggregates suspended prior to injection. It has also been shown that the sedimentation method with the injection attachment 100 reduces the variability of the administered dose. Specifically, it has been shown that the shape of the injection attachment 100 and the sedimentation method allow for increased precision. With increased precision, the dead volume can be overcome by applying a compensation factor in order to accurately administer the dose (i.e., the correct number of cells). The percentage improvement is as Figure 6 shown, with the dose falling within 40 - 90% of the expected range and a recovery rate of less than 20% compared to the surgeon's targeted dose. Thus, the cannula and method of the present invention can allow for the delivery of a complete dose of a cell composition.

[0066] In a particular aspect, the "sedimentation" method of the present invention prevents cells from being trapped in the cannula, thus precisely delivering the target dose. Specifically, in the sedimentation method of the present invention, the dose is flicked to resuspend the cells, such as aggregates, and then aspirated into a pre-prepared syringe attached to the cannula, thus entering the device through the same cannula used to transplant the cells back into the injection site (such as the subretinal space). In a specific aspect, the syringe attached to the cannula loaded with the dose is held vertically for about 2 - 15 minutes to allow the cells (such as cell aggregates) to settle before injecting the dose.

[0067] Thus, in certain embodiments, provided herein are a device and a method of using the device that enable the reliable delivery of a cell composition by providing an injection attachment 100 having a funnel-shaped hub 110. In a particular aspect, the present disclosure provides a cell delivery device that allows for reliable dose delivery of a cell composition (e.g., a composition of single cells or aggregated cells). In some aspects, the injection attachment 100 of the present invention allows for increased precision in dose delivery and further increased precision by implementing a dose compensation factor. Thus, in certain embodiments, provided herein are methods for delivering cell therapies (such as aggregated iPSC-derived PRP cell therapy).

[0068] In addition to improved dose consistency, in the method of the present invention, the injection accessory 100 described herein allows for a reduction in the dead volume of the cell solution relative to prior methods. For example, the injection accessory 100 as described herein may allow 50 ul of injectable cell solution to be aspirated into the cannula to deliver a 50 ul dose, whereas prior cannulas would require aspiration of 200 μL of injectable cell solution to deliver a 50 ul dose.

[0069] Furthermore, in certain aspects, the injection accessory 100 of the present invention includes a smaller diameter (i.e., larger gauge) compared to standard cannulas. Specifically, prior devices included cannulas with a larger diameter (e.g., 31G). The larger cannulas have been used to reduce shear forces during loading and injection. However, this study shows that cannulas with a smaller diameter (e.g., 33G) do not negatively impact cell compositions, such as cell aggregate compositions, contrary to the prior expectation that a smaller diameter would result in cell damage or loss. Specifically, it has been shown that the initial shear force on the cell composition does not affect the cell composition, particularly for the funnel-shaped hub of the injection accessory 100 of the present invention. Additionally, the smaller diameter cannulas provide the advantageous aspect of minimizing the amount of damage at the injection site and providing better surgical outcomes with less backflow of the cell composition.

[0070] In a particular aspect, the hub of the present invention is substantially free of capture points that would increase shear or turbulence as the cells move through the hub. The hub is generally smooth as the cells pass through the hub and into the cannula shaft. The reduction in turbulent shear stress helps to improve cell viability, while capture points can lead to cell waste and damage. Thus, the structure of the injection accessory 100 of the present invention allows for a reduction in cell loss.

[0071] Thus, in some aspects, the device of the present invention includes an injection accessory 100 that is coupled to a drug delivery mechanism 200, such as and A vision system or a manual syringe. The injection accessory 100 includes a funnel-shaped hub 110, which prevents any sedimentation or entrapment of cells in the hub, allowing for the delivery of higher doses of cells to a subject. The cannula tip 121 can be a 33-gauge or 31-gauge cannula tip. The method also 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 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 aspects, the hub 110 can include an internal volume 117 greater than 5 μL, 10 μL, 25 μL, 50 μL, 75 μL, 100 μL, 150 μL, 175 μL, or 200 μL.

[0072] The cell composition can contain a variety of excipients (e.g., buffers, salts, polymers, proteins, and preservatives), such as those used to stabilize cells or provide physiological osmotic pressure. The cells can be suspended in a formulated buffer. The buffer can be phosphate, citrate, acetate, or other organic acid buffers. The buffer can contain DMSO, benzonase, or albumin. In certain aspects, the buffer is a balanced salt solution (BSS), such as a BSS containing DMSO or albumin (especially human serum albumin (HSA)), such as 0.11%, 0.2%, 0.3%, 0.4%, or 0.5% HSA buffer. The cells can be resuspended, for example, by vortexing or manually flicking the vial containing the cell composition prior to loading. In some aspects, as Figure 17AAs shown, after loading and before injection, 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. In some aspects, the cells in the composition are allowed to settle in the cannula for, for example, more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes after loading and before injection. In some aspects, the cells in the composition are allowed to settle in the cannula for, for example, less than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes after loading and before injection. In some aspects, the cells in the composition are allowed to settle in the cannula for, for example, about 1 - 3, 2 - 5, 3 - 6, 4 - 7, 5 - 8, 6 - 9 or 7 - 10 minutes after loading and before injection. In some aspects, the cells in the composition are allowed to settle in the cannula for, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes after loading and before injection. In some aspects, the cells in the composition are allowed to settle in the cannula for, for example, at least 1 - 3, 2 - 5, 3 - 6, 4 - 7, 5 - 8, 6 - 9 or 7 - 10 minutes after loading and before injection.

[0073] Previously, a single - pass method has been used, which involves loading a cell composition through a first larger needle such as an 18G needle and then replacing the larger - diameter needle with a smaller - diameter needle such as a 31G cannula before injection. Generally, the larger - diameter needle is used for loading to reduce shear forces during loading, and the smaller - diameter needle is used for injection to reduce 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 help remove air from the syringe and cannula. Once the air is removed, that volume is dispensed until a lower dose (e.g., 50 μL) remains. The single - pass method requires needle replacement, which results in loss and disruption of the integrity of the cell composition, as well as variability in the administered dose.

[0074] However, in certain embodiments, the method involves a two - pass method, where the needle is not replaced between loading and injection. Current research shows that aggregates can pass through the cannula twice without causing corresponding damage ( Figure 9 ). This two - pass method also allows for a thorough inspection of syringe manipulation before dose delivery. In this two - pass method, cells are loaded and expelled through the same cannula, which reduces the number of manipulation steps and also reduces the chances of contamination and needlesticks. In particular, in the two - pass method, the same volume of dose (e.g., 50 μL) can be loaded and injected through a single needle compared to the initial large volume used in the single - pass method.

[0075] Accordingly, in certain embodiments, provided herein are delivery devices and delivery methods for delivering cell therapies such as PRP cell aggregates. The methods of the invention allow for the delivery of a consistent high dose of cells in a lower dose volume without significant cell loss. In a particular aspect, a cell therapy can be delivered subretinally to the eye of a subject via the device of the invention, the device comprising a cannula having a funnel-shaped hub. In some aspects, a bubble is inserted into the retina prior to the administration of the cells.

[0076] II. Definitions

[0077] The term “purified” does not require absolute purity; rather, it is intended to be 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, substantially free of other cell types.

[0078] As used in the specification, “a” or “an” can mean one or more. As used herein in the claims, when used in conjunction with the word “comprising,” the words “a” or “an” can mean one or more than one.

[0079] Unless expressly indicated to refer to alternatives only or that the alternatives are mutually exclusive, the use of the term “or” in the claims is used to mean “and / or,” although the present disclosure supports definitions that refer only to alternatives and “and / or.” As used herein, “another” can refer to at least a second or more.

[0080] The term “substantially” should be understood to mean that a method or composition includes only the specified steps or materials, and those steps or materials that do not materially affect the basic and novel characteristics of these methods and compositions.

[0081] As used herein, a composition or medium that is “substantially free” of a specified substance or material contains ≤30%, ≤20%, ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of the substance or material.

[0082] The terms “substantially” or “about” as used herein can be used to modify any quantitative comparison, numerical value, measurement, or other representation that can vary without resulting in a change in the basic function associated therewith.

[0083] The term “about” generally refers to within the standard deviation of the stated value, which is determined using standard analytical techniques for measuring the value. These terms can also refer to plus or minus 5% of the stated value.

[0084] As used herein, with respect to a specified component, "substantially free" means that no specified component is intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from any inadvertent contamination of the composition is far less than 0.05%, preferably less than 0.01%. Most preferably, the composition is one in which the amount of the specified component is undetectable by standard analytical methods.

[0085] The term "stem cell" as used herein refers to a cell that is capable of differentiating into multiple specialized cell types under appropriate conditions and that is capable of self-renewing and remaining in a substantially undifferentiated pluripotent state under other appropriate conditions. The term "stem cell" also encompasses pluripotent cells, multipotent cells, progenitor cells, and precursor cells. Exemplary human stem cells can be obtained 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 genital tissue. Exemplary pluripotent stem cells can also be generated from somatic cells by reprogramming the somatic cells into a pluripotent state by expressing certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs".

[0086] The term "pluripotency" refers to the property of a cell to differentiate into all other cell types in an organism except embryonic or placental cells. Even after long-term culture, pluripotent stem cells are still capable of differentiating into cell types of all three germ layers (e.g., ectodermal, mesodermal, and endodermal cell types). Pluripotent stem cells are embryonic stem cells derived from the inner cell mass of the blastocyst. In other embodiments, the pluripotent stem cells are induced pluripotent stem cells obtained by reprogramming somatic cells.

[0087] The term "differentiation" refers to the process by which unspecialized cells become more specialized types with changes in their structure and / or function. Mature cells typically have altered cell structure and tissue-specific proteins.

[0088] As used herein, "undifferentiated" refers to cells that display the characteristic markers and morphological features of undifferentiated cells, which clearly distinguish them from terminally differentiated cells of embryonic or adult origin.

[0089] An "embryoid body (EB)" is an aggregate of pluripotent stem cells that can differentiate into cells of the endoderm, mesoderm, and ectoderm. When pluripotent stem cells are aggregated under non-adherent culture conditions and thus form EBs in suspension, spherical structures are formed.

[0090] "Isolated" cells have been substantially separated or purified from other cells in an organism or culture. The purity of the isolated cells can be, for example, at least 99%, at least 98%, at least 95%, or at least 90%.

[0091] "Embryo" means a cell mass obtained by one or more divisions of a fertilized egg or an activated oocyte having a nucleus with artificial reprogramming.

[0092] "Embryonic stem (ES) cells" are undifferentiated pluripotent cells that are obtained from an embryo at an early stage, such as the inner cell mass at the blastocyst stage, or are generated by artificial means (e.g., nuclear transfer), and can give rise to any differentiated cell type in an embryo or an adult, including germ cells (e.g., sperm and eggs).

[0093] "Induced pluripotent stem cells (iPSCs)" are cells generated by reprogramming somatic cells by the combination of expression factors (referred to herein as reprogramming factors) or inducing their expression. 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 Oct 3 / 4), Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed into pluripotent stem cells by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors.

[0094] "Allele" means one of two or more forms of a gene. A diploid organism, such as a human, has two copies of each chromosome and thus carries one allele on each chromosome.

[0095] The term "homozygous" is defined as having two identical alleles at a particular locus. The term "heterozygous" means having two different alleles at a particular locus.

[0096] "Haplotype" refers to a combination of alleles at multiple loci along a single chromosome. A haplotype can be based on a set of single nucleotide polymorphisms (SNPs) on a single chromosome and / or alleles in the major histocompatibility complex.

[0097] As used herein, the term "haplotype-matched" is defined as a cell (e.g., an iPS cell) and the subject being treated sharing one or more major histocompatibility locus haplotypes. The haplotype of a subject can be readily determined using assays well known in the art. Haplotype-matched iPS cells can be autologous or allogeneic. Autologous cells that are grown and differentiated into PRP cells in tissue culture are inherently haplotype-matched to the subject.

[0098] "Substantially identical HLA type" means that the human leukocyte antigen (HLA) type of the donor matches that of the patient to the extent that when transplanted into the patient, the transplanted cells, which have been obtained by inducing the differentiation of iPSCs from somatic cells of the donor, can be transplanted.

[0099] "Super donor" as used herein refers to an individual who is homozygous for certain MHC class I and class II genes. These homozygous individuals can serve as super donors, and their cells, including tissues and other materials containing their cells, can be transplanted into individuals who are homozygous or heterozygous for that haplotype. A super donor can be homozygous for HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ locus / locus alleles, respectively.

[0100] "Feeder-free" or "independent of feeder cells" as used herein refers to a culture supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF) as an alternative to a feeder cell layer. Thus, "feeder-free" or feeder cell-independent culture systems and media can be used to culture and maintain pluripotent cells in an undifferentiated and proliferative state. In some cases, feeder-free cultures utilize an animal matrix (e.g., MATRIGEL TM ) or grow on a matrix such as fibronectin, collagen, or vitronectin. These methods allow human stem cells to remain in a substantially undifferentiated state without the need for a mouse fibroblast "feeder layer"

[0101] "Feeder layer" is defined herein as a coating layer of cells such as on the bottom of a culture dish. Feeder cells can release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, can attach.

[0102] When used in connection with a culture medium, extracellular matrix, or culture conditions, the term "defined" or "fully defined" refers to a culture medium, extracellular matrix, or culture conditions in which the chemical composition and amount of almost 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. Typically, a defined medium comprises a basal medium (e.g., Dulbecco's Modified Eagle 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 TM Medium.

[0103] For a culture medium, extracellular matrix, or culture system used with human cells, the term "Xeno-Free (XF)" means a condition in which the materials used are not of non-human animal origin.

[0104] "Pre-confluent" refers to a cell culture in which the proportion of the culture surface covered by cells is about 60 - 80%. Typically, pre-confluent refers to a culture in which about 70% of the culture surface is covered by cells.

[0105] The term "neural retinal progenitor cell" or "NRP" refers to a cell whose differentiation potential is limited to neural retinal cell types.

[0106] The term "photoreceptive" cell or "PR" cell refers to a cell within the photoreceptor lineage (i.e., mature) pathway, which includes early and late markers of photoreceptive cells (rods, cones, or both), before and after upregulation of the expression of either rhodopsin (rods) or one of the three cone opsins (cones).

[0107] The term "photoreceptor precursor cell" or "PRP" refers to a cell differentiated from embryonic stem cells or induced pluripotent stem cells, which can differentiate into a photoreceptive cell expressing either rhodopsin or one of the three cone opsins. The photoreceptor can be a rod and / or cone photoreceptor.

[0108] The term "retinal degeneration-related disease" refers to any disease caused by congenital or acquired retinal degeneration or abnormalities. Examples of retinal degeneration-related diseases include retinal dysplasia, retinal degeneration, age-related macular degeneration, Stargardt disease, Best disease, choroideremia, hereditary macular degeneration, myopic degeneration, RPE tear, macular hole, diabetic retinopathy, retinitis pigmentosa, hereditary retinal disease or degeneration, hereditary macular degeneration, cone-rod dystrophy, rod-cone dystrophy, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, and retinal trauma.

[0109] As used herein, the term "ocular disorder" refers to a disease, disorder, discomfort or injury in the ocular region. In some embodiments, the ocular disorder may include disorders of the posterior segment of the eye. In other embodiments, the ocular disorder may include disorders of the anterior segment of the eye. The ocular disorder may be related to retinal epithelial cells and / or photoreceptor cells. The ocular disorder may be related or associated with diabetes (e.g., diabetic macular edema, retinal arterial 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, serpignous choroiditis, uveitis syndrome, cytomegalovirus retinitis or endophthalmitis), genetics (e.g., Coat's disease or familial exudative vitreoretinopathy) or cancer (e.g., posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, combined hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative tumor, retinal astrocytoma, retinal carcinoma or intraocular lymphoid tumor). The disease may be a retinopathy, such as acute and chronic macular neuroretinopathy, or central serous chorioretinopathy.For example, eye disorders can 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 placoid pigment epitheliopathy, Behcet's disease, birdshot retinochoroidopathy, posterior uveitis, posterior scleritis, serous choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal arterial occlusive disease, central retinal vein occlusion, diffuse intravascular coagulation lesions, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal arterial microaneurysms, Coats' disease, parafoveal telangiectasia, hemi-retinal vein occlusion, papillophlebitis, carotid artery disease (CAD), frosted branch angitis, sickle cell retinopathy, angioid streaks, familial exudative vitreoretinopathy, Eales' disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor-related retinal diseases, congenital hypertrophy of the retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastases, combined hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative tumors, retinal astrocytoma, intraocular lymphoid tumors, myopic retinal degeneration, acute retinal pigment epitheliitis, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal carcinoma, and any combination thereof. Eye injuries can include injuries caused by abrasions, stabs, scratches, penetrating wounds, perforating wounds, or intraocular foreign bodies.

[0110] As used herein, "therapeutically effective amount" refers to an amount of a compound that is sufficient to effect such treatment when administered to a subject to treat a disease or disorder.

[0111] "Inducer" is defined herein as a molecule that regulates gene expression such as activating a gene within a cell. An inducer can bind to a repressor or an activator. An inducer acts by inhibiting a repressor.

[0112] As used herein, the term "transplanted" bilayer refers to transplanted cells that migrate into the host retina and form pre- and post-synaptic mechanisms such that the transplanted cells and host cells are ready to form synapses.

[0113] As used herein, the term "biodegradable" refers to a material that provides initial structural support for the delivered cells, but degrades over time into products that are non-toxic to the transplanted host and do not cause morbidity at the donor site.

[0114] 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 a transgenic species thereof. In certain embodiments, the patient or subject is a primate, particularly a human. Non-limiting examples of human patients are adults, adolescents, infants, and fetuses.

[0115] As used herein, "smooth" means that the cannula hub is substantially free of protrusions projecting inwardly from the inner hub surface.

[0116] III. Cell Delivery Device

[0117] In some aspects, the present disclosure provides a device for delivering a cell composition. The delivery device may include an injection attachment having a cannula and a funnel-shaped hub, such as Figures 1 - 2 the injection attachment 100 in Figure 1 The cannula located at the distal end 112 of the hub 110 in Figure 1 may have an outer diameter of less than 0.3 mm, such as an outer diameter 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. The cannula hub 110, such as Figure 1 the distal end 112 in Figure 1a tapered design (such as having an outer diameter in the range of about 0.30 mm to 0.18 mm or less). The cannula tip 121 can have an outer diameter of about 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 in diameter), 31-gauge, 32-gauge, 33-gauge, 34-gauge (e.g., 0.18 mm in diameter), 35-gauge, 36-gauge, 37-gauge, 38-gauge (e.g., 0.12 mm in diameter), 39-gauge, or 40-gauge (e.g., 0.10 mm in diameter) cannula tip, particularly a 30-34 gauge cannula tip with a diameter of about 0.30 mm to about 0.18 mm. For example, the cannula 120 can be a 25-gauge cannula 120 with a 33-gauge cannula tip 121. Additionally, the cannula tip 121 can be made of 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, flexible, and extendable.

[0118] The injection accessory 100 can be attached to a syringe 220, such as a syringe that allows controlled microinjection, such as a MICRODOSE TM syringe. The injection accessory 100 can include a coupling mechanism 113, such as a Luer lock hub, or a connection to the syringe 220, the extension tube 230, or the infusion line 240. The tubing can be silicone tubing, such as silicone tubing. The device can also include a viscous fluid control package 210. In other aspects, the tube 230 can be packaged with the syringe 220, such as a MICRODOSE TM syringe, without the viscous fluid control package 210.

[0119] Embodiments of the present disclosure include methods of use involving cannulas. Now referring to Figures 1 - 2 the injection accessory 100 shown includes a funnel-shaped hub 110, a cannula 120, and a cannula tip 121. Figure 1 A side view of the injection accessory 100 is shown, while Figure 2 a partial cross-sectional view taken along line Figure 1 2-2 is shown.

[0120] In an exemplary embodiment, the coupling mechanism 113, such as a Luer lock hub, can be coupled to a drug delivery mechanism 200 (not shown), including, for example, a vision system or a manual syringe. It should be understood that the listed drug delivery mechanisms are merely examples of specific embodiments, and according to other embodiments of the present disclosure, any suitable drug delivery mechanism can be coupled.

[0121] Now referring to Figure 2, in the illustrated embodiment, the funnel-shaped hub 110 includes an internal volume 117 defined by a proximal end 111 (referred to herein, and in the applications claiming priority to the present disclosure, as the first end), a distal end 112 (referred to herein, and in the applications claiming priority to the present disclosure, as the second end), and an inner surface 115. The term "funnel-shaped" as used herein can be broadly defined as the internal volume 117 that is configured to direct particles (e.g., cells suspended in a fluid) through the hub 110 into the cannula 120 while minimizing or eliminating the presence of residual particles. In a specific embodiment, the proximal end 111 may include a threaded portion (e.g., coupling mechanism 113) or other suitable coupling mechanism configured to engage a drug delivery mechanism (including, for example, a "Luer lock"-type design). The coupling mechanism 113 may include an inner surface of an outer surface configured to couple with the drug delivery mechanism 200. The coupling mechanism 113 may include, for example, angled protrusions that are received by corresponding protrusions on the drug delivery mechanism 200 to couple by threaded engagement. The coupling mechanism 113 and the drug delivery mechanism 200 may be coupled by snap fit, glue, epoxy, welding, or other engagement sufficient to prevent accidental separation of the coupling mechanism 113 from the drug delivery mechanism 200. During use, the drug delivery mechanism may transfer contents from the internal volume 117 through the cannula 120 and cannula tip 121 into a subject, as Figure 17B shown.

[0122] As Figure 2 shown, the inner surface 115 may be generally funnel-shaped. The funnel shape helps to minimize residual particles 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 may include a constant angle between the proximal end 111 and the second end 122. As Figure 2 shown, the taper may include a variable angle between the proximal end 111 and the second end 122. In the illustrated example, the inner diameter D1 (measured on the inner surface 115 within the internal volume 117) gradually decreases as measured from the proximal end 111 towards the distal end 112. Although Figure 2The two sides shown are mirror images of each other. It is contemplated that the inner surface 115 may taper towards one side, or may taper inwards as a whole, but may taper inwards and outwards 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 way, the inner surface 115 does not include any protrusions or other features that may limit the contents of the internal volume 117 from leaving the hub 110 and entering the cannula 120. Thus, the contents of the internal volume 117 (e.g., cell suspension, therapeutic agent, or other suitable contents) can move unrestrictedly from the proximal end 111 of the injection attachment 100 to the distal end 112, and then further into the cannula 120 and the tip 121. The method and the injection attachment 100 can result in at least 99% of the cells contained in the injection attachment 100 being injected into the 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 in the injection attachment 100 are injected into the tissue.. The continuous tapering of the inner surface 115 and the absence of protrusions in the internal volume 117 also allow the contents of the internal volume to be thoroughly mixed before administration to a subject.

[0123] In certain embodiments, the cannula 120 (referred to herein, and in the applications claiming priority to the present disclosure, as the rod) can be configured such that the diameter of the rod does not constrict or limit the injection dose provided by the injection attachment 100. In certain embodiments, the cannula 120 can be configured as a 25-gauge cannula or a rod of other suitable diameter. In certain embodiments, the cannula tip 121 can be configured as a 33-gauge (33G) cannula, a 31-gauge (31G) cannula, or a tip of other suitable diameter. Thus, the combination of the needle hub 110 (having an inner surface 115 without protrusions or restrictions) and the suitable diameter of the cannula 120 allows the injection attachment 100 to provide thorough mixing of the contents of the internal volume 117 and administration of the contents, without restrictions in the internal volume 117. Thus, the contents of the internal volume 117 can be administered to a subject in an accurate and reproducible dose.

[0124] IV. Cell Delivery Methods

[0125] In certain aspects, the present disclosure provides methods for cell transplantation, such as cell aggregate or single cell suspension therapy. The methods of the invention can be applied to various cell transplantation procedures known in the art. The transplantation can include providing a volume of cells to a cell delivery device (not shown) that includes a proximal end and a distal end opposite the proximal end. The device can include an internal volume configured to receive the cell volume.

[0126] The device may include a cannula (e.g., cannula 120) extending from a funnel-shaped hub 110. The cannula may include a cannula tip 121. The cannula tip 121 may be a 33-gauge cannula tip. In some embodiments, the cannula tip 121 is a 32-gauge cannula tip, 31-gauge cannula tip, 30-gauge cannula tip, 29-gauge cannula tip, 28-gauge cannula tip, 27-gauge cannula tip, 26-gauge cannula tip, 25-gauge cannula tip, 24-gauge cannula tip, 23-gauge cannula tip, or 22-gauge cannula tip.

[0127] The funnel-shaped hub 110 is coupled to the distal end of the device and is in fluid communication with the internal volume. The funnel-shaped hub 110 may include a proximal end 111 and a distal end 112, wherein an inner surface 115 extends 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.

[0128] The diameter of the inner surface 115 at the distal end 112 may be less than the inner wall diameter at the proximal end 111. The inner surface 115 may taper between the proximal end 111 and the distal end 112. The cannula (e.g., cannula 120) may be coupled to the proximal end 111 of the funnel hub 110. The cannula 120 is coupled to the distal end 112 using glue 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 Figure 2 shown, the cannula 120 is coupled to the distal end 112 such that no portion of the cannula 120 enters the internal volume 117.

[0129] The device may be oriented such that the distal end is below the proximal end relative to the ground for a period of time. This period of time is sufficient for the cells to settle at the distal end of the hub 110. Sedimentation may refer to approximately 90% of the cells settling within the hub 110, as Figure 17A shown. In some embodiments, sedimentation means 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 means between 50% and 90% of the cells, between 55% and 85% of the cells, between 60% and 80% of the cells, or between 65% and 75% of the cells.

[0130] This period of time is sufficient for the cells to sediment at the distal end 112 of the funnel-shaped hub 110. As Figures 17A - 17B shown, the cells (C) may sediment at the distal end 112 of the funnel-shaped hub 110. Figures 17A - 17BAlso shown is a liquid solution (S) in which the cells C are suspended. The funnel-shaped hub 110 may comprise a transparent material to allow visual inspection of the cells sedimented therein. The time period may be between 30 seconds and 10 minutes. The time period 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. The time period 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, a centrifugal force may be applied to the hub to reduce the time required for the cells to sediment near the distal end of the hub.

[0131] The method may include the step of inserting a cannula into a subject's tissue. The cannula tip 121 may be blunt-ended and configured to enter the tissue through an incision. In some embodiments, the cannula tip 121 may be pointed and configured to pierce the tissue to enter. The tissue may be retinal tissue.

[0132] As Figure 17B shown, when injecting cells into a subject's tissue, the device may be held at an angle (α). During the injection, the device may be held at an angle A of about 45 degrees relative to the ground. During the injection, the device may be held at an angle A of about 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 the injection process, the device may be held at an angle A between 20 degrees and 70 degrees relative to the ground. During the injection process, the device may be held at an angle A between 25 degrees and 65 degrees, between 30 degrees and 60 degrees, between 35 degrees and 55 degrees, between 40 degrees and 50 degrees, or between 45 degrees and 55 degrees relative to the ground.

[0133] The method may include the step of injecting a volume of cells into a subject's tissue using a cell delivery device. The volume of cells may be provided to the internal device and injected into the subject's tissue through the same injection attachment 100. For example, the cannula tip 121 and the cannula 120 are not replaced between providing a volume of cells into the internal volume and injecting the cells into the subject's tissue.

[0134] In some aspects, the present disclosure provides methods for delivering cell therapies, such as cell aggregate or single cell suspension therapies. The methods of the invention can be applied to a variety of cell therapies known in the art. Delivery can include microsurgery using a microinjection cannula or similar device, such as ophthalmic surgery. Delivery can include subretinal injection by slow, controlled injection of the cell therapy. The cannula can be coupled to a syringe or system that allows a predetermined uniform injection pressure (psi), such as between 0 psi and 30 psi (e.g., greater than 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 psi, or psi of 0-30, 2-28, 4-26, 6-24, 5-20, 15-25, 20-25 or 25-30 or psi of 0-15, 0-16, 0-17, 0-18, 0-19 or 0-20), and the injection pressure can be controlled by a foot pedal 250 that controls the pressure in a variable manner. The cells can be stored at a suitable temperature, such as 4°C.

[0135] In certain embodiments, the delivery device of the present invention provides a mechanism for delivery to the eye (such as subretinal administration), or a mechanism for delivering a therapeutic agent to the mammalian eye (e.g., the posterior segment of the eye), more particularly the human eye, and a method for treating and / or preventing eye disorders and / or diseases, particularly retinal / choroidal disorders or diseases, by such subretinal administration of such a therapeutic agent. Such methods provide a mechanism for treating various 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 methods can also be used to treat / prevent a variety of 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; rubeosis iritis inflammatory diseases, chronic posterior uveitis and panuveitis, tumors, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting following a combined vitrectomy and lensectomy, vascular diseases, retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, neovascularization of the optic nerve, diabetic macular edema, cystoid macular edema, macular edema, retinitis pigmentosa, retinal vein occlusion, proliferative vitreoretinopathy, angioid streaks, and retinal artery occlusion, and neovascularization resulting from a perforated globe or ocular trauma. The methods of the present invention can also be used to treat ocular symptoms caused by diseases or disorders that have both ocular and non-ocular symptoms.

[0136] In some embodiments, the preparation and delivery of the cell composition includes the steps of thawing and resuspending the cell composition in a vial (e.g., a 50 mL conical tube). The cell composition can be formulated in a buffer as described herein. The cell composition can be centrifuged, such as 1, 2, 3, 4, 5 times or more (e.g., in the formulation buffer). For clinical applications, a master dose of the cell composition can be prepared in a formulation buffer (e.g., 25 - 50, 30 - 75, 40 - 80, 50 - 90, or 60 - 100 μL of buffer). At this stage, the cell composition can be dissociated and counted. Next, a master dose can be prepared to achieve the target concentration of the cell composition. Finally, the dose can be aliquoted (e.g., 50 μL) into appropriate vials. For animal studies, such as in rodents, the cell composition can be dissociated and counted before centrifuging the remaining volume. The master dose can be prepared by aspirating the buffer, resuspending the pellet with the remaining supernatant, measuring the volume, and adding the formulation buffer to achieve the target concentration. Then the dose can be aliquoted (e.g., 15 μL).

[0137] The device of the present invention can be used for transplantation, such as cell rescue therapy or whole tissue replacement therapy. Certain embodiments can use the device of the present invention to enhance the maintenance and repair of eye tissue (for any desired disorder, including retinal degeneration or severe injury). Retinal degeneration may be associated with age - related macular degeneration (AMD), hereditary macular degeneration, Stargardt macular dystrophy, Best disease, choroideremia, hereditary retinal degeneration (including retinitis pigmentosa, cone / rod and rod / cone dystrophies), diabetic retinopathy, retinal vascular diseases, injury caused by retinopathy of prematurity (ROP), viral infections of the eye, and other retinal / ocular diseases or injuries / traumas.

[0138] On the other hand, the present invention provides a method of treating an individual in need, comprising transplanting a composition comprising cells such as PRP cells into the individual. The composition can be administered to the eye, such as into the sub - retinal space. These individuals may suffer from hereditary macular or retinal degeneration, such as retinitis pigmentosa, cone / rod or rod / cone dystrophies, Stargardt disease, Best disease, choroideremia, retinal dysplasia, retinal degeneration, diabetic retinopathy, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, injury caused by retinopathy of prematurity (ROP), or other retinal trauma or injury.

[0139] The devices and cells described herein can be used to manufacture medicaments for treating the conditions of patients in need. The cells can be cryopreserved in advance. In some aspects, the photoreceptors disclosed are derived from iPSCs and can thus be used to provide "personalized medicaments" for patients with eye diseases. In some embodiments, somatic cells obtained from a patient can be genetically engineered to correct a disease-causing mutation and differentiated into PRPs. Alternatively, iPSCs generated from healthy donors or HLA-homozygous "super donors" can be used.

[0140] The methods and compositions of the invention can treat or prevent a variety of eye disorders. Such disorders include retinal diseases or conditions generally associated with retinal dysfunction or degeneration, retinal damage, and / or loss of retinal pigment epithelium and / or photoreceptors. Disorders that can be treated include, but are not limited to, retinal degenerative diseases such as Stargardt macular dystrophy, retinitis pigmentosa, rod / cone and cone / rod dystrophy, macular degeneration (such as age-related macular degeneration, myopic macular degeneration, or other acquired or hereditary macular degeneration), retinal damage caused by retinopathy of prematurity (ROP), and diabetic retinopathy. Other disorders include Leber congenital amaurosis, hereditary or acquired macular or retinal degeneration, Best disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, other dystrophies of photoreceptor cells, and retinal damage resulting from injury caused by any of light, laser, inflammation, infection, radiation, neovascularization, or trauma. In certain embodiments, methods are provided for treating or preventing disorders characterized by retinal degeneration, comprising administering to a subject in need an effective amount of a composition comprising PRP by the device of the invention.

[0141] In some embodiments, the cell delivery device and method of use thereof of the present invention 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 method for treating and / or preventing eye disorders and / or diseases (particularly retinal / choroidal disorders or diseases) by such subretinal administration of such a therapeutic agent. Such methods provide a mechanism for treating various diseases and / or disorders of a 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 a treatment / prevention method can also be used to treat / prevent a variety of 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; rubeosis iridis, iritis, inflammatory diseases, chronic posterior uveitis, and panuveitis, tumors, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting from vitrectomy combined with lens removal, vascular diseases, retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, neovascularization of the optic nerve, diabetic macular edema, cystoid macular edema, macular edema, retinitis pigmentosa, retinal vein occlusion, proliferative vitreoretinopathy, angioid streaks, and retinal artery occlusion, and neovascularization resulting from globe perforation or ocular trauma. The method of the present invention can also be used to treat ocular symptoms caused by diseases or disorders that have both ocular and non-ocular symptoms.

[0142] The delivery method of the present invention can be used for cell transplantation in various cell therapies, such as for treating peripheral arterial disease, myocardial infarction, stroke, Parkinson's disease, Huntington's disease, 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 of the present invention can be used to transplant cells into any soft tissue, including but not limited to the eye, heart, kidney, liver, tumor, or muscle. The dose range of cells can be from several hundred to several million cells.

[0143] V. Cell Composition

[0144] The terms "population of cells", "cell composition", "cell therapy composition", or "composition of cells" are used interchangeably herein and refer to a group of cells that are generally of the same type. A population of cells can be derived from a common progenitor cell or can contain more than one cell type. An "enriched" population of cells refers to a population of cells derived from a starting population of cells (e.g., an unfractionated heterogeneous population of cells) that contains a higher percentage of a particular cell type than the percentage of that cell type in the starting population of cells. A population of cells can be enriched for one or more cell types and depleted of one or more cell types.

[0145] A cell composition can comprise a single cell suspension or cell aggregates. The composition of the cells can be autologous or allogeneic. The composition of the cells can include or be derived from PSCs, such as iPSCs or hESCs. The composition of cells can include one cell type (i.e., a single cell) or two or more cell types (i.e., a multicellular). In some aspects, the term "cell" as used herein refers to a composition of cells. In some aspects, the cell composition can comprise an organoid or be a whole tissue substitute.

[0146] In some aspects, the cell aggregates can include cell clusters. A cell cluster can include at least 2 cells. In some embodiments, the cell cluster includes more than 3, more than 4, more than 5, more than 10, more than 15, more than 20, more than 25, or more than 30 cells. In some embodiments, the cell cluster includes at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 cells. The diameter of a single cell is about 9 μm. In some embodiments, a single cell is about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, about 10 μm, about 10.5 μm, or about 11 μm. The cell composition can include a distribution of cell clusters of different sizes therein.

[0147] In some aspects, 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 and can be used for drug testing as well as the research and treatment of diseases. PSCs can differentiate into any one of the 216 cell types found in adult organisms, such as neurons, cardiomyocytes, smooth muscle cells, osteocytes, hepatocytes, keratinocytes, insulin-producing cells, hematopoietic cells, and endothelial cells. In certain aspects, the PSC-derived cells of the present invention can comprise one or more of the 216 cell types. In some aspects, the cell composition to be transplanted includes an engineered stem cell-derived tissue or organ composed of multiple cell types. The main cell types derived from the endoderm include hepatocytes and insulin-producing cells. Mesodermal progenitor cells 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 tissues, and obtain all types of blood cells, respectively. Cells differentiated into the ectodermal lineage include cells of the epidermis, external sensory organs, central and peripheral nervous systems, such as functional neurons that can be used to treat neurodegenerative diseases (such as acute spinal cord injury).

[0148] In some aspects, the cell composition can be genetically engineered to knockout and / or knockin genes. This can be carried out by various genome editing methods, including CRISPR technology, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALEN) technology.

[0149] The cell therapy composition can also include other agents. For example, the composition can include agents that further protect or stabilize the cells to be transplanted. In certain embodiments, the composition contains vitamins, minerals, antioxidants, cryoprotectants, viscosity enhancers, coenzymes, membrane stabilizers, lipids, carbohydrates, hormones, growth factors, anti-inflammatory agents, polynucleotides, proteins, peptides, alcohols, organic acids, organic small molecules, etc.

[0150] The cell composition may comprise a pharmaceutically acceptable excipient, including any solvent, dispersion medium, diluent, or other excipient suitable for the particular formulation desired herein. Liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) is used for formulating pharmaceutical compositions. Various available excipients and their known preparation techniques are disclosed. Any conventional excipient that would produce any undesired biological effect on the substance or its derivatives, or interact with any other one or more components of the pharmaceutical composition in a detrimental manner, is not used unless otherwise indicated. Thus, its use is intended to be within the scope of the present invention.

[0151] In some embodiments, the purity of the pharmaceutically acceptable excipient is at least 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the US Food and Drug Administration. In some embodiments, the excipient is of pharmaceutical grade. In some embodiments, the excipient complies with the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopoeia, and / or International Pharmacopeia standards.

[0152] Pharmaceutically acceptable excipients for producing cell therapy compositions include, but are not limited to, inert diluents, dispersants, surfactants and / or emulsifying agents, disintegrants, preservatives, buffering agents, 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, dibasic calcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, examples include mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, etc. and combinations thereof.

[0153] Exemplary buffers include, but are not limited to, citrate buffers, acetate buffers, phosphate buffers, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium grubionate, calcium glucoceptate, calcium gluconate, D - gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxidephosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, primary potassiumphosphate, potassium hydrogen phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, phosphoric acid, sodium bicarbonate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen - free water, isotonic saline, Ringer's solution, ethanol, and the like, and combinations thereof.

[0154] In additional embodiments, kits for cell transplantation are provided herein. The kit can include a buffer for cell washing, a device for cell washing, cells, a syringe, a needle, a cup, a container, an alcohol swab, an anesthetic, an antibiotic, an antioxidant, vitamins, lipids, carbohydrates, hormones, growth factors, and the like. In certain embodiments, the kit includes a cannula that includes a funnel - shaped hub, a tube, and a pneumatic syringe, such as a MICRODOSE TM syringe. In certain embodiments, the components of the kit are sterilized and packaged for ease of use by a surgeon or other healthcare professional or for use in the manufacturing process. The kit can also include instructions for using the cannula and other agents during the transplantation. The kit can provide the components needed for single - use. The kit package can also include the packaging materials and information required by government regulatory agencies for the administration of drugs and / or medical devices.

[0155] VI. Eye cells

[0156] In some embodiments, retinal progenitor cells (RPE), photoreceptor cells, and / or photoreceptor precursor cells are delivered by the devices provided herein. The cells in the retina that are directly sensitive to light are photoreceptor cells. Photoreceptors are light - sensitive neurons on the outer part of the retina and can be rod cells or cone cells. During the process of light conduction, photoreceptor cells convert the incident light energy focused by the cornea and lens into electrical signals, which are ultimately sent to the brain through the optic nerve. Vertebrates have two types of photoreceptor cells, including cone cells and rod cells. Cone cells are adapted to detect fine details, central, and color vision and function well in bright light. Rod cells are responsible for peripheral and dim - light vision. The nerve signals from rod cells and cone cells are processed by other neurons in the retina.

[0157] 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 methods such as immunocytochemistry, western blot analysis, flow cytometry, or enzyme-linked immunosorbent assay (ELISA). For example, a 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, when evaluated by quantitative RT-PCR, the expression of these genes in photoreceptors is as low as approximately 1 / 1000 to 1 / 100 in ES cells or iPSC cells.

[0158] Photoreceptor markers can be detected at the mRNA level, for example, by reverse transcription polymerase chain reaction (RT-PCR), western blot analysis, microarray, or RNA sequencing (including single-cell RNA sequencing dot blot analysis using sequence-specific primers in standard amplification methods). The expression of tissue-specific markers detected at the protein or mRNA level is considered positive if it is at least or approximately 2, 3, 4, 5, 6, 7, 8, or 9 times, and more particularly more than 10, 20, 30, 40, 50 times or higher, than that of control cells such as undifferentiated pluripotent stem cells or other irrelevant cell types. Dysfunction, damage, and loss of photoreceptor cells are factors in many eye 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 hereditary retinal diseases and acquired retinal dysfunction, diseases, and injuries. A potential treatment for such diseases is the transplantation of PRP and / or PR into the retinas of people in need of such treatment. It is hypothesized that supplementing PRP and / or PR through their transplantation can delay, halt, or reverse degeneration, improve retinal function, and prevent blindness caused by such afflictions. However, obtaining PRP and / or PR directly from human donors and embryos is challenging.

[0159]

[0160] ​The retinal pigment epithelium acts as a barrier between the blood flow and the retina and interacts closely with photoreceptors in maintaining visual function and choroidal blood supply. The retinal pigment epithelium consists 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; light absorption and prevention of photooxidation; re-isomerization of all-trans retinol to 11-cis retinal; phagocytosis of shed photoreceptor membranes; and secretion of various factors crucial for the structural integrity of the retina.

[0161] Mature retinal pigment epithelium expresses markers such as cellular retinaldehyde-binding protein (CRALBP), RPE65, bestrophin gene (VMD2), and pigment epithelium-derived factor (PEDF). Dysfunction of the retinal pigment epithelium is associated with many vision-altering disorders such as retinal pigment epithelial detachment, dysplasia, atrophy, retinopathy, retinitis pigmentosa, macular dystrophy or degeneration (including age-related macular degeneration).

[0162] 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 identified by their cobblestone morphology and the initial appearance of pigment. In addition, the differentiated RPE cell layer has a transepithelial resistance / TER and generates a transepithelial electrical potential / TEP across the monolayer (TER > 100 ohms.cm2; TEP > 2 mV), transports fluids, lactate, and CO2 from the apical side to the basal side, and regulates the polarized secretion of cytokines.

[0163] RPE cells express several proteins that can serve as markers for detecting their identity and maturation status by methods such as immunocytochemistry, western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). For example, RPE-specific markers may 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, when evaluated by quantitative RT-PCR, the expression of these genes in RPE cells is as low as approximately 1 / 1000 - 1 / 100 of that in ES cells or iPSC cells.

[0164] RPE cell markers can be detected at the mRNA level, for example, by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analysis, or dot blot analysis (using publicly available sequence data and sequence-specific primers in standard amplification methods) for such detection. Expression of a tissue-specific marker detected at the protein or mRNA level is considered positive if it is at least or about 2, 3, 4, 5, 6, 7, 8, or 9-fold, and more particularly more than 10, 20, 30, 40, 50-fold or higher, of that of control cells such as undifferentiated pluripotent stem cells or other irrelevant cell types.

[0165] Dysfunction, injury, and loss of RPE cells are factors in many eye diseases and conditions, including age-related macular degeneration (AMD), hereditary macular degeneration (including Best disease, Stargardt disease, and choroideremia), and other forms of hereditary retinal diseases and acquired retinal dysfunction, diseases, and injuries (including but not limited to RPE tears / ruptures). A potential treatment for such diseases is transplantation of RPE cells into the subretinal space of a person in need of such treatment. It is hypothesized that replenishing RPE cells by their transplantation can delay, halt, or reverse degeneration, improve retinal function, and prevent blindness caused by such diseases. However, obtaining RPE cells directly from human donors and embryos is challenging.

[0166] In some aspects, RPE cells are generated from iPSCs, such as by the methods disclosed in PCT / US2016 / 050543 and PCT / US2016 / 050554.

[0167] In some embodiments, methods are provided for generating photoreceptors from a substantially single-cell suspension of PSCs (such as human iPSCs). In some embodiments, the PSCs are cultured to pre-confluence. In certain aspects, the PSCs are dissociated by incubating them with a cell dissociation solution or enzyme (such as Versene, trypsin, AACCUTASE TM or TRYPLE TM ). The PSCs can also be dissociated into a substantially single-cell suspension by pipetting.

[0168] In addition, bryostatin (e.g., about 2.5 μM) can be added to the culture medium to increase the survival of PSCs after dissociation into single cells while the cells do not adhere to the culture vessel. A ROCK inhibitor rather than bryostatin can alternatively be used to increase the survival of PSCs after dissociation into single cells.

[0169] Once a single-cell suspension of PSCs is obtained, the cells are typically seeded in a suitable culture vessel (such as a tissue culture plate, such as a flask, multi-layer flask, 6-well, 12-well, 24-well, 96-well, or 10 cm plate). Culture vessels for culturing one or more cells may include but are not particularly limited to: flasks, flasks for tissue culture, culture dishes, Petri dishes, culture dishes for tissue culture, multidishes, microplates, microwell plates, multiplates, multiwell plates, micro slides, chamber slides, test tubes, trays, chambers, culture bags, and roller bottles, provided that they are capable of culturing stem cells therein. Depending on the needs of the culture, the cells can be cultured at 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 derivable therefrom. In certain embodiments, the culture vessel can be a bioreactor, which can refer to any ex vivo device or system that supports a bioactive environment to enable cell propagation. 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 therefrom.

[0170] In some aspects, PSCs (such as iPSCs) are plated at a cell density suitable for efficient differentiation. Typically, the cells are plated at a cell density of about 1,000 to about 75,000 cells / cm 2 , such as about 5,000 to about 40,000 cells / cm 2 . In a 6-well plate, the cells can 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, such as about 50,000 cells per well.

[0171] PSCs, such as iPSCs, are typically cultured on culture plates coated with one or more cell adhesion proteins to promote cell adhesion 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 the culture surface as a means of providing a solid support for pluripotent cell growth. The term "extracellular matrix (ECM)" is well recognized in the art. Its components can include, but are not limited to, one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, nestin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, ankyrin, chondronectin, syndecan, osteopontin, osteocalcin, bone sialoprotein, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Other ECM components can 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 culture plates coated with vitronectin. In some embodiments, the cell adhesion protein is a human protein.

[0172] The extracellular matrix protein can be of natural origin and purified from human or animal tissues, or, the ECM protein can be a genetically engineered recombinant protein or a natural synthesis. The ECM protein can be a complete protein or in the form of a natural or engineered peptide fragment. Examples of ECM proteins that can be used for cell culture substrates include laminin, type I collagen, type IV collagen, fibronectin, and vitronectin. In some embodiments, the matrix composition is free of heterologous protein components. For example, in a matrix free of heterologous protein components for culturing human cells, matrix components of human origin can be used, where any non-human animal components can be excluded.

[0173] In some aspects, the total protein concentration in the matrix composition can be from about 1 ng / mL to about 1 mg / mL. In some preferred embodiments, the total protein concentration in the matrix composition is from about 1 μg / mL to about 300 μg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is from about 5 μg / mL to about 200 μg / mL.

[0174] Cells can be cultured with nutrients necessary to support the growth of each specific cell population, such as photoreceptors or PSCs. Generally, cells are cultured in a growth medium containing a carbon source, a nitrogen source, and a buffer to maintain pH. The medium can also contain fatty acids or lipids, amino acids (such as non-essential amino acids), one or more vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, pH indicators, and inorganic salts. Exemplary growth media include minimal basal media supplemented with various nutrients (such as non-essential amino acids and vitamins), such as Dulbecco's Modified Eagle Medium (DMEM) or ESSENTIAL 8 TM (E8 TM ) medium to enhance stem cell growth. Examples of minimal basal media include, but are not limited to, Minimal Essential Medium Eagle (MEM) Alpha medium, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640 medium, Medium 199, and F12 medium. In addition, the minimal basal medium can be supplemented with additives such as horse serum, calf serum, or fetal bovine serum. Alternatively, the medium can be serum-free. In other cases, the growth medium can contain a "knockout serum replacement", referred to herein as a serum-free preparation, which is optimized to grow and maintain undifferentiated cells, such as stem cells, in culture. KNOCKOUT TM Serum replacement is disclosed, for example, in U.S. Patent Application No. 2002 / 0076747, which is incorporated herein by reference. Preferably, PSCs are cultured in a medium with fully defined components and without feeder cells.

[0175] Thus, single-cell PSCs are typically cultured in a medium with fully defined components after plating. In some aspects, about 18 - 24 hours after seeding, the medium is aspirated and fresh medium such as E8 TM medium is added to the culture. In some aspects, single-cell PSCs are cultured in a medium with fully defined components for about 1, 2, or 3 days after plating. Preferably, single-cell PSCs are cultured in a medium with fully defined components for about 2 days before the differentiation process.

[0176] In some embodiments, the medium can contain or not contain any replacement for serum. Replacements for serum can include materials appropriately containing albumin (such as lipid-rich albumin, albumin replacements such as recombinant albumin, plant starches, dextrans, and proteolytic products), transferrin (or other iron transport proteins), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiol glycerol, or equivalents thereof. The replacement for serum can be prepared by the methods disclosed, for example, in International Publication No. WO98 / 30679. Alternatively, for greater convenience, any commercially available materials can be used. Commercially available materials include NOCKOUT TMSerum replacement (KSR), chemically defined concentrated lipids (Gibco), and GLUTAMAX TM (Gibco).

[0177] Other culture conditions can be appropriately defined. For example, the culture temperature can be from about 30°C to 40°C, such as at least or about 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, but is not particularly limited thereto. In one embodiment, the cells are cultured at 37°C. The CO2 concentration can be from about 1% to 10%, such as from about 2% to 5%, or any range derivable therefrom. The oxygen tension can be at least, up to, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20% or any range derivable therefrom. VII. EXAMPLES

[0178] The following examples are included to illustrate the preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention and thus can be considered to constitute a preferred mode for its practice. However, based on the present disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention and still obtain the same or similar results.

[0179] Example 1 - Delivery of Photoreceptor Precursors Aggregates

[0180] Studies have been conducted to develop a dosing delivery procedure for large animals and clinical settings. Allogeneic human induced pluripotent stem cell (iPSC)-derived photoreceptor precursor (iPRP) aggregates have been developed as a cell-based PR replacement therapy. After dose preparation (REP-00798), the iPRP aggregates were transplanted using Alcon’s Constellation vision system [1], an ophthalmic microsurgery system. The MICRODOSE TM injection kit 1 mL syringe (MedOne Surgical, Inc., #3275) [2] was attached to the vision system (Alcon) through a tube in a viscous fluid control (VFC) package (#8065750957). Studies have been conducted to determine the most suitable dose loading and delivery procedures, including generating and testing the geometries of novel cannula hubs for delivering cell products such as aggregated cell products.

[0181] Dose formulation. Unless otherwise stated, to prepare iPRP aggregates for transplantation, the cells are thawed and resuspended in BSS with 0.2% HSA buffer. Subsequently, multiple centrifugation and washing steps are performed, and samples are taken, dissociated with 10X TrypLE and cell counted. Using the viable cell concentration, a dose formulation ( Figure 3 ) is prepared.

[0182] Dose analysis. For bench tests, the dose is either loaded into a 1 mL syringe (BD #309628 or MedOne Surgical, Inc., #3275) through an 18G needle and then expelled through a 31G (MedOne #3218) or 33G (MedOne #3262) cannula (single-pass method), or loaded into the syringe through a 31G (MedOne #3218) or 33G (MedOne #3262) cannula and then expelled through the same cannula (double-pass method). Manual pressure is generated with a plunger (BD #309628) to draw up and expel the dose, or the pressure is generated by attaching a 1 mL syringe (MedOne Surgical, Inc., #3275) to the vision system via a viscous fluid control (VFC) pack (#8065750957).

[0183] Once expelled, to analyze the number of recovered cells, the aggregates are enzymatically dissociated in 10X TrypLE for 30 minutes, quenched in DMEM / F12 + B27 + Benzonase (iPRP quenching medium), triturated and counted. Alternatively, to obtain the integrity of the aggregates, the aggregates are transferred to a Multisizer 4 and passed through a 280 μm aperture.

[0184] Overview of the iPRP polymerization device structure. To deliver iPRP aggregates to the subretinal space of large animals or to humans in the clinic, a vision system (Alcon, Figure 4 ) [1] is used to load and inject the volume under controlled pressure. The MICRODOSE TM syringe kit 1 mL syringe (MedOne Surgical, Inc., #3275) [2] is attached to the vision system through a tube in a viscous fluid control (VFC) package (#8065750957). The injection attachment 100 (MedOne Polytip cannula, 25G / 33G) is connected to the syringe, through which a 50 μL volume of cells is delivered into the subretinal space via a pre-determined uniform injection pressure (psi), which is controlled by a foot pedal.

[0185] Comparison of 31G cannulas versus 33G cannulas. The integrity of the iPRP aggregates is negatively affected by their handling. Thus, to reduce shear forces during dose loading and injection, larger diameter cannulas can be used. However, because the cannula must be inserted through a retinal incision to deliver the dose behind the retina, smaller cannulas may be required to minimize the amount of damage to the retina. Additionally, larger retinotomies (which allow smaller gauge cannulas) create wider holes in the retina, which increases the incidence of the injected iPRP aggregates flowing back out with the bubble and subsequently delivering less product. Historically, 31Ga cannulas have been used, but due to potentially positive effects on the surgery, this study investigated whether smaller diameter cannulas (33G) would have a negative impact on the aggregate product.

[0186] To test the effect of cannula gauge on aggregate integrity, iPRP aggregates were thawed and prepared. 200 μL of the final aggregated product formulation was manually loaded into a syringe through an 18G needle. The needle was then replaced with a MedOne 31G (#3218) or 33G cannula (#3262). Entrapped air was removed from the syringe and cannula, and the unused portion of the dose was expelled until the plunger reached the 50 μL mark (the volume of the clinical dose). Then 50 μL of the dose was expelled and the aggregate size was analyzed on a Multisizer (280 μm aperture). The gate was pulled from approximately 17 μm to 168 μm to capture the aggregates, and the volume percentage of the product falling within the gate was measured. Additionally, the median aggregate diameter was calculated within the gate. Based on these results, in terms of aggregate integrity, using a 33G cannula should be comparable to a 31G cannula, as there were no significant differences in aggregate volume or median diameter. Since the effect of cannula diameter on aggregate integrity was negligible and using a smaller cannula was beneficial for the surgery, the 33G cannula (#3262) was further investigated.

[0187] Study of variable surgical dose delivery. Because iPRP consists of aggregates, it quickly settles out of the formulation buffer (REP-00798). This affects not only the dose formulation but also creates problems during dose delivery. Thus, tests were conducted to determine whether settling of the product in the syringe would result in inaccurate dosing.

[0188] Historically, in vitro experiments during this period used a manual 1 mL syringe attached to an 18G needle during loading (200 μL), removed the needle, and expelled the cells through a 33G cannula (50 μL). Then, 50 μL of the injected iPRP aggregate dose was dissociated in 10X TrypLE and counted. It should be noted that the experiments described herein were initially designed to study different 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 since the vehicle had no significant effect, the data were combined for this study. Results are shown as the percentage 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 live cells recovered by the target dose ( Figure 6 , blue circles). After eight experiments with a total of 33 individual injections over four batches (iPRP0045 20200727 - CD133_60, iPRP0047A, iPRP0047B, and iPRP0049A), the dose was within approximately 40% to 90% of the expected range.

[0189] Around this time, a video of a mock surgery using the cell product of the present invention by a surgeon and a surgeon's assistant was provided. From this experiment, very few cells were recovered after injection. When viewing the video, it was noted that the surgical procedure was slow. Additionally, importantly, not only was the time increased, but the tip of the cannula was oriented upward for most of the time, which may have caused the aggregates to settle towards the syringe plunger, preventing them from being injected. To test this hypothesis, three doses of iPRP0047A were produced, one dose for each test condition. The control was performed in the same manner as the first 8 experiments, where the dose was raised and injected rapidly (FCDI standard; Figure 6, dashed line). Specifically, for the FCDI standard, the cell aggregate composition was resuspended in BSS containing 0.2% HSA. The Constellation vision system was connected to the tubing, viscous fluid control pack, and microinjection kit syringe. The syringe and cannula were perfused with buffer, with the cannula facing up, and the foot pedal was depressed to clear excess air and BSA + HSA. The syringe was vertically inserted into the clamp on the 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. Once the foot pedal was depressed, the Constellation system was then set to inject at a pressure of 10 PSI. For the second dose, the scientists noted any changes in syringe orientation, and the surgeon was aware of the changes in syringe orientation based on the video generated by the duration of dose uptake during injection (surgeon's approach; Figure 6 , square). Specifically, the surgeon's orientation included keeping the dose perpendicular to the ground during settlement and injecting at an angle A of approximately 45 degrees as expected, as Figure 17B shown. To determine if the time in the syringe reduced dose recovery, for the third case, the syringe was loaded and then held for the same length of time (approximately 5 minutes) as the surgeon manipulated the dose, but kept parallel to the ground and rolled back and forth between the scientist's fingers with the aim of keeping the aggregates suspended ( Figure 6 , diamond).

[0190] Once collected, all iPRP aggregate injection doses were dissociated in 10X TrypLE and counted to calculate the percentage of expected cell recovery. The surgeon's orientation dose recovery rate was very prominent, with a recovery rate of less than 20% (injection #35; Figure 6 , pink square), while the FCDI standard dose (injection #34, Figure 6 , green dashed line) and the 5-minute roll dose (injection #36, Figure 6 , light green diamond) had typical recovery rates. These findings emphasize the need for 1) extensive training of surgeons and surgical assistants to maintain the aggregates in suspension, or 2) a change in the delivery method.

[0191] Flicking the loaded syringe is sufficient to keep the aggregates suspended. Given that iPRP aggregates may fall out of the formulation buffer once loaded into the syringe, resulting in an inaccurate dose to be injected, attempts were made to determine ways to maintain proper aggregate distribution in the syringe. Three different experiments were conducted using batch iPRP0046C. The dose was prepared such that 2 million cells were expected to be delivered from the injection. Using Constellation, 200 μL was loaded into the syringe through an 18G needle. After replacing the 18G needle with a 33G cannula (MedOne #3262), air was cleared and additional volume was expelled such that the plunger reached the 50 μL mark. Typically, at this step in the protocol, the dose remains in the device for no more than 2 minutes before injection. To simulate a potential "worst-case" scenario, the syringe was placed on its side for 5 minutes, at which point it was evident that the aggregates had settled to the side of the syringe. After this waiting period, the dose was either expelled immediately (without mixing) or first mixed by vortexing or flicking the syringe. A total of 14 injections were performed in these three experiments. As expected, mixing before expelling the dose from the syringe resulted in significantly higher recovery ( Figure 7 ). Based on these results, it was determined that flicking was sufficient to redistribute the dose.

[0192] Utilize aggregate sedimentation by using injection accessory 100. Flick the syringe loaded with iPRP aggregates to redistribute the sedimented aggregates to provide a mixed formulation and a reliable injection ( Figure 7 ). However, despite these changes, concerns about surgical injection variability remained, so different dose delivery methods were tested, including purposefully causing the aggregates to settle within the hub of the cannula.

[0193] Hypothetically, if a 50 μL dose is loaded into a syringe and all the aggregates settle vertically into the cannula hub, when injecting this dose, all the aggregates should be expelled first, and fewer aggregates may be trapped along the side of the syringe or return to the plunger due to sedimentation and inappropriate resuspension techniques. Therefore, next, it was tested whether this new sedimentation method improved the aggregate recovery rate compared to the previous suspension method. In this experiment, for the "resuspended" dose, iPRP aggregates were prepared. As in the previous experiment, a 200 μL dose was manually aspirated into the syringe through an 18G needle. Then the 18G needle was replaced with a 33G cannula (MedOne #3262), the air and extra volume were removed, leaving a 50 μL dose in the device. Then this 50 μL dose was injected into a test tube for analysis. However, for the study of vertical sedimentation, a 33G cannula was attached to the syringe, and both were perfused with a vehicle (BSS + HSA) to ensure no air was trapped in the cannula or syringe. Once the air was cleared, the piston was pushed back to 0 μL. Then 50 μL of the dose was withdrawn through the cannula while keeping the device in the vertical orientation (cannula down), and a 5-minute waiting time was started. After the incubation time ended, this 50 μL dose was also injected into a test tube for downstream analysis. Then the cells were separated and counted to determine the delivered dose. Unfortunately, although the suspension method provided an expected recovery of approximately 2 million cells, the syringe in which the cells settled injected less than 25% of the expected dose ( Figure 8A ).

[0194] When carefully observing the cannula hub, the reason for this lack of cell recovery becomes obvious. The cannula hub provides a space to trap cells as designed (Figure 8B, arrow). Even after the syringe plunger has been fully depressed, a ring of white aggregates can still be seen in the cannula hub 301 ( Figure 8C , arrow).

[0195] Therefore, the injection attachment 100 was developed, in which the hub would be shaped like a funnel ( Figure 8D ), such that compared to an off-the-shelf cannula (MedOne #3262, Figure 8B ), there is limited or no space to trap aggregates. Then experiments were conducted with this new cannula design, hoping that the sedimentation method could improve the consistency of the injected dose compared to the suspension method.

[0196] Returning to the same experimental design as Figure 8A performed, an off-the-shelf 33G cannula (#3262) using the suspension method was compared with the injection attachment 100 using aggregate sedimentation. In the first experiment iPRP0046C, a dose of iPRP aggregates was prepared and 7 injections were performed using the injection attachment 100 after the sedimentation method, while 5 injections were performed using the off-the-shelf cannula after the suspension method (Figure 8E )。In exactly the same manner, using the same iPRP aggregates, the following experiments were conducted with 4 injections using the injection attachment 100 and 5 off-the-shelf cannula injections( Figure 8F )。Using the injection attachment 100 with the sedimentation method resulted in CVs of 9.2% and 8.4% respectively, while the off-the-shelf cannulas showed CVs of 29.2% and 23.4% respectively. This reduced variability in the injection attachment 100 and the sedimentation method eliminated the need to keep the aggregates in suspension, thereby alleviating concerns about proper surgical training for the correct delivery of the iPRP aggregates.

[0197] Note that in the "resuspension" method, the aggregates enter the cannula through an 18G needle and exit through a 33G cannula, while the "sedimentation method" allows the aggregates to pass through a 33G cannula and exit through a 33G cannula. Since the integrity of the aggregates can be affected by shear stress, the integrity of the aggregates passing through the cannula one or two times was investigated.

[0198] A first experiment was conducted to test the possible effect of passing through the cannula twice on the integrity of the aggregates. The iPRP aggregates were thawed and a dose was prepared. As described above for the suspension method, the dose was loaded and injected, except that for testing the double pass through the cannula, a 200 μL dose was loaded into the syringe through a 33G cannula instead of an 18G needle. After injection, a 50 μL dose was analyzed on a Multisizer using a 280 μm aperture. The gate was opened from approximately 17 μm to 168 μm, and the volume percentage of the product falling within the gate, as well as the median aggregate diameter within the gate, were measured. The median aggregate diameter was found to be approximately 40 - 60 μm, such as 40 - 45 μm, 45 - 55 μm, 50 - 55 μm, or 50 - 60 μm. Although both measurements decreased slightly when the aggregates were passed through the 33G cannula twice, the effect was small enough to continue using the sedimentation method( Figure 9 )。

[0199] Dose variability was calculated. 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, which would ensure that the concentration of the bulk was deliberately higher than the final target concentration. Samples were then taken from the bulk dose and dissociated to obtain cell counts. The bulk volume was measured and then aliquoted into multiple tubes and diluted with additional BSS + HSA to reach the appropriate concentration. These volumes (referred to as the master dose) were then aliquoted into doses for injection( Figure 10 )。

[0200] To determine the variability between master doses, 28 master doses from 10 experiments were compared using seven different batches of iPRP aggregate products (iPRP0046C, iPRP0047B, iPRP0049C, iPRP0049D, iPRP0054F, iPRP0055A, iPRP0057). To standardize reporting, the percentage of the expected cell concentration (actual counted concentration divided by the target concentration) was plotted. The results showed that these master doses contained on average 93% of the expected cell concentration, with a coefficient of variation of 11.4%( Figure 11 ). 80% of the doses were within ±16% of the target cell concentration, 90% were within ±23% of the target dose, 100% were within ±28% of our dose, with a minimum expected cell concentration of 73% and a maximum expected cell concentration of 111%( Figure 11 ).

[0201] In these studies, one source of error that could contribute to differences was the preparation of multiple concentrations of master doses from a single donor preparation. However, this variability exists in clinical or large animal GLP studies because the protocol only requires one master dose concentration. For these studies, the donor was prepared and samples were taken for dissociation and cell counting to calculate the donor concentration. However, with the exception of 20 μL, the entire volume of the donor was transferred to a new tube and diluted to the target concentration (master dose), rather than dividing that volume among multiple tubes. The master dose was then aliquoted into injection doses( Figure 12 ).

[0202] Experiments generating one master dose from one donor concentration produced lower variability than when generating multiple master doses from a donor. Nine master doses prepared from 5 different batches of iPRP aggregates showed an average master dose of 93.5% of the expected cell concentration, with a coefficient of variation of 5%. 88% of the doses were within ±10% of our target cell concentration, with a minimum expected cell concentration of 84% and a maximum expected cell concentration of 99%( Figure 13 ).

[0203] Since the aggregates sediment rapidly, variability may be introduced when aliquoting the master dose into aliquots. In nine experiments using iPRP, 5 to 12 aliquots of the aggregates were dispensed, each containing 60 - 70 μL or 210 μL (this excess volume allowed each aliquot to contain 50 μL or 200 μL of counted cells after sampling so that they could be used for downstream injection experiments). A 5 μL sample was taken from each aliquot, dissociated and counted to determine the concentration of the aliquot dose.

[0204] As described above, the percentage of the expected cell concentration was used for this data because these experiments targeted different dose concentrations. Importantly, while variability was observed between experiments, the variability between doses within an experiment showed a coefficient of variation ranging from approximately 7.5% to 15.5%.

[0205] Dose compensation factor. The new device loading (50 μl dose through the cannula) procedure set in the injection attachment 100 provided improved precision, but further studies were conducted to evaluate the dosing precision. In multiple studies, it was observed that, according to the calculated dose concentration, fewer injected cells were recovered than expected. Therefore, the dose compensation factor was considered.

[0206] Using Constellation, doses of 1.4 to 3.4 million cells were prepared and injected in 11 experiments with a total of 82 injections using 6 different batches of iPRP aggregates (iPRP0047B, iPRP0049C, iPRP0049D, iPRP0054F, iPRP0055A, and iPRP0057). Extrapolating a linear fit line from all data points yielded the best fit equation y = 0.9255x - 0.5777( Figure 15A ). Using this equation, doses of 1.7 million and 2.8 million loaded cells were calculated to achieve the target doses of 1 million (clinical) and 2 million (non-human primate) injected cells, respectively. For doses of 3 million and 4 million (clinical) cells, a linear fit line was extrapolated from all data points generated for 391 individual injections, which covered 2 iPSC lines, 8 iPRP batches (16 sub-batches) of all 4 doses, and the best fit equation y = 0.0348x - 0.1239 was determined( Figure 15B ). Using this equation, doses of 4.5 million and 5.95 million cells were calculated to be loaded to achieve the target doses of injecting 3 million and 4 million cells, respectively. The test results for the 1M, 2M, 3M, and 4M dose targets can be seen in Figure 15C .

[0207] In 6 experiments with a total of 25 injections using 3 batches (iPRP0047B, iPRP0055A, and iPRP0057), the variability of the injected clinical dose compensated with 1.7 million cells was tested. The average dose was 1.04 million cells, and the coefficient of variation (CV) was 19%. 80% of the doses were within ±27% of the target 1 million dose, 90% were within ±33% of our dose, 100% fell within ±42% of the dose, with a minimum dose of 0.64 million injected cells and a maximum dose of 1.41 million injected cells( Figure 15B)。For a non-human primate target of 2 million cells, a calculated dose of 2.8 million cells was generated. In 15 injections across 3 experiments and 4 batches (iPRP0047B, iPRP0054F, iPRP0055A, and iPRP0057), the average dose was 2.1 million cells with a CV of 16%. 80% of the doses were within ±19% of our target dose of 1 million, 90% were within ±23% of our dose, 100% fell within ±38% of our dose, the minimum dose injected was 1.23 million cells, and the maximum dose injected was 2.45 million cells (15C).

[0208] Observations and improvements in large animals (Figure 16) and clinical iPRP aggregate dose delivery have been described above. The data indicate that with the implemented changes, a more reliable dose can be delivered per injection.

[0209] The initial protocol required loading cells through an 18G needle and then replacing that needle with a 31G cannula prior to injection. While this protocol was effective, there was room for improvement. The 31G cannula was sufficient to deliver cells subretinally; however, it required a large retinotomy with a greater likelihood of retinal damage and the possibility of iPRP aggregate reflux. Testing of a 33G cannula with a smaller diameter showed that the increased shear stress on the aggregates had a minimal effect on the aggregate size distribution. Therefore, further studies were conducted using the 33G cannula.

[0210] Subsequent testing determined whether the aggregates could pass through the cannula twice without attendant damage. Since no effect on aggregate integrity was observed, the dose loading was converted to a 33G cannula, replacing the 18G needle. Reducing the handling steps not only made the process easier but also reduced the chances of contamination and needlestick injuries.

[0211] This two-pass method also allowed for a thorough inspection of the syringe operation prior to dose delivery. The one-pass method required loading a 200 μL dose into the syringe to help clear air from the syringe and cannula. Once the air was removed, volume was dispensed until a 50 μL dose remained. To prevent aggregate settling, both the loading process and the time between loading and delivery needed to be done quickly with this method, and even then, variability in the cells delivered during the procedure was seen. For this reason, allowing the aggregates to settle was purposefully studied in hopes of injecting the most consistent dose.

[0212] Although allowing aggregates to settle towards the cannula tip initially did not work due to the geometry of the cannula hub, the injection attachment 100 design using the new hub (which acts more like a funnel) provides a more consistent dose. With this new cannula design, a 50 ul dose can be loaded into the cannula, allowed to settle towards the tip for 5 minutes, and then injected, eliminating the need to keep the aggregates suspended prior to injection. In addition to improving dose consistency, it should be noted that the dose used in this method is one quarter of the original method (50 μL vs. 200 μL).

[0213] For example, the subject technology is described in terms of various aspects described below. For convenience, various examples of aspects of the subject technology are described in numbered fashion (1, 2, 3, etc.). These are provided as examples and do not limit the subject technology. Note that any dependent aspects can be combined in any combination and placed in their own independent aspects, such as aspect 1 or aspect 18. Other aspects can be presented in a similar manner.

[0214] Aspect 1. A method for cell transplantation, comprising injecting cells into a subject's tissue using an injection attachment, the injection attachment including a cannula having a funnel-shaped hub.

[0215] Aspect 2. The method of aspect 1, wherein the length of the cannula hub is less than 30 mm.

[0216] Aspect 3. The method of aspect 1 or 2, wherein the cannula includes a cannula tip of 30 gauge or smaller.

[0217] Aspect 4. The method of aspect 3, wherein the cannula tip is blunt.

[0218] Aspect 5. The method of aspect 3, wherein the cannula tip is sharp.

[0219] Aspect 6. The method of aspect 5, wherein the cannula tip is further defined as a cannula tip.

[0220] Aspect 7. The method of aspect 3, wherein the cannula tip is a 34-gauge, 33-gauge, 32-gauge, 31-gauge, or 30-gauge cannula tip.

[0221] Aspect 8. The method of aspect 3, wherein the cannula tip is a 33-gauge cannula tip.

[0222] Aspect 9. The method of any of aspects 3-8, wherein the cells are loaded into the cannula using a cannula tip of 30 gauge or smaller.

[0223] Aspect 10. The method of any of aspects 3-8, wherein the cells are loaded into the cannula using a 33-gauge cannula tip.

[0224] Method 11. The method according to any one of Methods 3 - 8, wherein the cells are loaded into the cannula and injected into the tissue of the subject using the same cannula tip.

[0225] Method 12. The method according to any one of Methods 1 - 11, wherein the cannula tip is not replaced between loading into the cannula and injection into the tissue of the subject.

[0226] Method 13. The method according to any one of Methods 1 - 12, wherein the cannula tip is made of a flexible polyimide material or metal.

[0227] Method 14. The method according to any one of Methods 1 - 13, wherein the cannula tip is flexible.

[0228] Method 15. The method according to any one of Methods 1 - 13, wherein the cannula tip is rigid.

[0229] Method 16. The method according to any one of Methods 1 - 7, wherein the hub includes a one - way check valve.

[0230] Method 17. The method according to any one of Methods 1 - 16, wherein the hub includes a coupling mechanism.

[0231] Method 18. The method according to Method 17, wherein the coupling mechanism is a Luer lock.

[0232] Method 19. The method according to Method 18, wherein the Luer lock is connected to a drug delivery mechanism.

[0233] Method 20. The method according to Method 19, wherein the drug delivery mechanism is a syringe.

[0234] Method 21. The method according to Method 20, wherein the syringe is a microinjection syringe.

[0235] Method 22. The method according to Method 20, wherein the syringe is further connected to a conduit.

[0236] Method 23. The method according to Method 22, wherein the conduit is connected to a pressure control system.

[0237] Method 24. The method according to any one of Methods 1 - 23, wherein the cells are delivered from the injection attachment under controlled pressure.

[0238] Method 25. The method according to any one of Methods 1 - 24, wherein the cells are not redistributed before loading into the injection attachment.

[0239] Method 26. The method according to any one of Methods 1 - 25, wherein the cells are redistributed before loading into the injection attachment.

[0240] Method 27. The method of Method 26, wherein the cells are redistributed by vortexing or manual agitation.

[0241] Method 28. The method of any one of Methods 1-27, wherein the cells are injected into the eye of the subject.

[0242] Method 29. The method of Method 28, wherein the cells are injected subretinally.

[0243] Method 30. The method of any one of Methods 1-29, wherein the cells are further defined as cell aggregates.

[0244] Method 31. The method of any one of Methods 1-29, wherein the cells are further defined as single cells.

[0245] Method 32. The method of any one of Methods 1-31, wherein the cells are in a formulation buffer.

[0246] Method 33. The method of Method 32, wherein the formulation buffer is a balanced salt solution.

[0247] Method 34. The method of Method 33, wherein the balanced salt solution further comprises benzonase and / or human serum albumin.

[0248] Method 35. The method of Method 30, wherein the cell aggregates are not suspended when injected into the tissue of the subject.

[0249] Method 36. The method of Method 35, wherein the cells are present in the cannula for at least 5 minutes between loading and injection to allow the aggregates to settle in the cannula hub.

[0250] Method 37. The method of Method 35, wherein the cells are present in the cannula for at least 5 minutes between loading and injection to allow the cells to settle in the cannula hub.

[0251] Method 38. The method of any one of Methods 35-37, wherein the cannula points downward to allow the cells to settle in the cannula hub.

[0252] Method 39. The method of any one of Methods 30-36, wherein the cell aggregates are photoreceptor progenitor cell aggregates.

[0253] Method 40. The method of any one of Methods 1-36, wherein the cells are retinal progenitor cells and / or photoreceptor progenitor cells.

[0254] Method 41. The method of any one of Methods 30-36, wherein the cell aggregates are retinal progenitor cells and / or photoreceptor progenitor cells.

[0255] Method 42. The method of any one of Methods 1 - 39, wherein the cells are injected in a volume less than 200 μL.

[0256] Method 43. The method of any one of Methods 1 - 42, wherein the cells are injected in a volume less than 100 μL.

[0257] Method 44. The method of any one of Methods 1 - 43, wherein the cells are injected in a volume of about 50 μL.

[0258] Method 45. The method of any one of Methods 1 - 44, wherein at least 1 million cells are injected.

[0259] Method 46. The method of any one of Methods 1 - 45, wherein at least 2 million cells are injected.

[0260] Method 47. The method of any one of Methods 1 - 46, wherein at least 25% of the cells loaded into the injection accessory are injected into the tissue of the subject.

[0261] Method 48. The method of any one of Methods 1 - 47, wherein at least 30% of the cells loaded into the injection accessory are injected into the tissue of the subject.

[0262] Method 49. A method for treating an eye disorder of a subject, comprising performing cell transplantation in the eye of the subject using an injection accessory, the injection accessory including a cannula having a funnel-shaped hub.

[0263] Method 50. The method of Method 49, wherein the length of the cannula hub is less than 30 mm.

[0264] Method 51. The method of Method 49 or 50, wherein the cannula includes a cannula tip of 30 gauge or smaller.

[0265] Method 52. The method of Method 51, wherein the cannula tip is blunt.

[0266] Method 53. The method of Method 51, wherein the cannula tip is sharp.

[0267] Method 54. The method of Method 53, wherein the cannula tip is further defined as a needle tip.

[0268] Method 55. The method of Method 51, wherein the cannula tip is a 34-gauge, 33-gauge, 32-gauge, 31-gauge, or 30-gauge cannula tip.

[0269] Method 56. The method of Method 51, wherein the cannula tip is a 33-gauge cannula tip.

[0270] Method 57. The method of any one of Methods 51 - 56, wherein the cells are loaded into the cannula using a cannula tip of 30 gauge or smaller.

[0271] Method 58. The method of any one of Methods 51 - 56, wherein the cells are loaded into the cannula using a 33 - gauge cannula tip.

[0272] Method 59. The method of any one of Methods 51 - 56, wherein the same cannula tip is used to load the cells into the cannula and inject them into the eye of the subject.

[0273] Method 60. The method of any one of Methods 49 - 59, wherein the cannula tip is not replaced between loading into the cannula and injecting into the eye of the subject.

[0274] Method 61. The method of any one of Methods 49 - 60, wherein the cannula tip is made of a flexible polyimide material or metal.

[0275] Method 62. The method of any one of Methods 49 - 61, wherein the cannula tip is flexible.

[0276] Method 63. The method of any one of Methods 49 - 61, wherein the cannula tip is rigid.

[0277] Method 64. The method of any one of Methods 49 - 55, wherein the hub includes a one - way check valve.

[0278] Method 65. The method of any one of Methods 49 - 64, wherein the hub includes a coupling mechanism.

[0279] Method 66. The method of Method 65, wherein the coupling mechanism is a Luer lock.

[0280] Method 67. The method of Article 66, wherein the Luer lock is connected to a drug delivery mechanism.

[0281] Method 68. The method of Method 67, wherein the drug delivery mechanism is a syringe.

[0282] Method 69. The method of Method 68, wherein the syringe is a microinjection syringe.

[0283] Method 70. The method of Method 68, wherein the syringe is further connected to a conduit.

[0284] Method 71. The method of Method 70, wherein the conduit is connected to a pressure control system.

[0285] Method 72. The method of any one of Methods 49 - 71, wherein the cells are delivered from the injection attachment under controlled pressure.

[0286] Method 73. The method of any one of Methods 49 - 72, wherein the cells are not redistributed before loading into the injection accessory.

[0287] Method 74. The method of any one of Methods 49 - 73, wherein the cells are redistributed before loading into the injection accessory.

[0288] Method 75. The method of Method 74, wherein the cells are redistributed by vortexing or manual agitation.

[0289] Method 76. The method of any one of Methods 49 - 76, wherein the cells are subretinally injected.

[0290] Method 77. The method of any one of Methods 49 - 76, wherein the cells are further defined as cell aggregates.

[0291] Method 78. The method of any one of Methods 49 - 76, wherein the cells are further defined as single cells.

[0292] Method 79. The method of any one of Methods 49 - 78, wherein the cells are in a formulation buffer.

[0293] Method 80. The method of Method 79, wherein the formulation buffer is a balanced salt solution.

[0294] Method 81. The method of Method 80, wherein the balanced salt solution further comprises benzonase and / or human serum albumin.

[0295] Method 82. The method of Method 77, wherein the cell aggregates are not suspended when injected into the tissue of the subject.

[0296] Method 83. The method of Method 82, wherein between loading and injection, the cells are present in the cannula for at least 5 minutes to allow the aggregates to settle in the cannula hub.

[0297] Method 84. The method of Method 82, wherein between loading and injection, the cells are present in the cannula for at least 5 minutes to allow the cells to settle in the cannula hub.

[0298] Method 85. The method of any one of Methods 82 - 84, wherein the cannula points downward to allow the cells to settle in the cannula hub.

[0299] Method 86. The method of any one of Methods 77 - 83, wherein the cell aggregates are photoreceptor progenitor cell aggregates.

[0300] Method 87. The method of any one of Methods 49 - 83, wherein the cells are retinal progenitor cells and / or photoreceptor progenitor cells.

[0301] Method 88. The method according to any one of methods 77 - 83, wherein the cell aggregate is a retinal progenitor cell and / or a photoreceptor precursor cell.

[0302] Method 89. The method according to any one of methods 49 - 86, wherein the cells are injected in a volume of less than 200 μL.

[0303] Method 90. The method according to any one of methods 49 - 89, wherein the cells are injected in a volume of less than 100 μL.

[0304] Method 91. The method according to any one of methods 49 - 90, wherein the cells are injected in a volume of approximately 50 μL.

[0305] Method 92. The method according to any one of methods 49 - 91, wherein at least 1 million cells are injected.

[0306] Method 93. The method according to any one of methods 49 - 92, wherein at least 2 million cells are injected.

[0307] Method 94. The method according to any one of methods 49 - 93, wherein at least 25% of the cells loaded into the injection attachment are injected into the eye of the subject.

[0308] Method 95. The method according to any one of methods 49 - 94, wherein the eye disorder is a hereditary retinal disease, age - related macular degeneration (AMD), hereditary macular degeneration, Stargardt 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.

[0309] Method 96. A cannula device for cell transplantation, comprising:

[0310] A tubular hub, wherein the tubular hub comprises:

[0311] An outer wall;

[0312] An inner wall;

[0313] A first end; and

[0314] A second end, wherein the first end has a funnel shape that slopes towards the second end.

[0315] Method 97. The device according to method 96, wherein the inner diameter of the tubular hub gradually decreases when measured from the first end towards the second end.

[0316] Method 98. The device according to method 96, wherein the hub is substantially free of capture points.

[0317] Variant 99. The device of Variant 96, wherein the hub has a smooth surface.

[0318] Variant 100. The device of Variant 96 or 97, wherein the length of the tubular hub is less than 30 mm.

[0319] Variant 101. The device of any one of Variants 96 - 100, wherein the first end is attached to a cannula tip.

[0320] Variant 102. The device of Variant 101, wherein the cannula tip is blunt.

[0321] Variant 103. The device of Variant 102, wherein the cannula tip is sharp.

[0322] Variant 104. The device of Variant 103, wherein the cannula tip is further defined as a needle tip.

[0323] Variant 105. The device of Variant 101, wherein the cannula tip is 30 gauge or smaller.

[0324] Variant 106. The device of Variant 105, wherein the cannula tip is a 34 - gauge, 33 - gauge, 32 - gauge, 31 - gauge, or 30 - gauge cannula tip.

[0325] Variant 107. The device of Variant 105, wherein the cannula tip is a 33 - gauge cannula tip.

[0326] Variant 108. The device of any one of Variants 96 - 106, wherein the second end includes a one - way check valve integrated in the hub, which prevents backflow.

[0327] Variant 109. The device of Variant 108, wherein the valve is further connected to a conduit.

[0328] Variant 110. The device of any one of Variants 101 - 109, wherein the cannula tip has an outer diameter in the range of about 0.30 mm to about 0.18 mm

[0329] Variant 111. The device of any one of Variants 101 - 110, wherein the cannula tip is made of a flexible polyimide material or metal.

[0330] Variant 112. The device of any one of Variants 101 - 111, wherein the cannula tip has a length of 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm.

[0331] Variant 113. The device of any one of Variants 101 - 112, wherein the cannula tip is flexible.

[0332] Variant 114. The device of any one of Variants 101 - 113, wherein the cannula tip is rigid.

[0333] Mode 115. The device of any one of Modes 101 - 114, wherein the cannula is attached to the injection device.

[0334] Mode 116. The device of any one of Modes 101 - 115, wherein the injection device is a syringe.

[0335] Mode 117. The device of Mode 116, wherein the syringe includes a handle for adjusting the fluid flow path.

[0336] Mode 118. The device of Mode 116 or 117, wherein the syringe is a microinjection syringe.

[0337] Mode 119. The device of any one of Modes 116 - 118, wherein the syringe includes a fluid volume of approximately 1 mL.

[0338] Mode 120. The device of any one of Modes 101 - 119, wherein the tubular hub includes a fluid volume of less than 200 uL.

[0339] Mode 121. The device of any one of Modes 101 - 119, wherein the tubular hub includes a fluid volume of less than 100 uL.

[0340] Mode 122. The device of any one of Modes 101 - 121, wherein the tubular hub includes a fluid volume of approximately 50 uL.

[0341] Mode 123. The device of any one of Modes 101 - 122, wherein the cannula device is further defined as a subretinal delivery device.

[0342] Mode 124. The device of any one of Modes 101 - 123, which is used for delivering cells to the tissue of a subject.

[0343] Mode 125. The device of any one of Modes 101 - 123 is used for treating an eye disorder of a subject, including administering an effective amount of cells to the eye of the subject.

[0344] Mode 126. The use of Mode 125, wherein the same cannula tip is used to load the cells into the cannula and inject them into the eye of the subject.

[0345] Mode 127. The use of Mode 125, wherein the cannula tip is not replaced between loading into the cannula and injecting into the eye of the subject.

[0346] Mode 128. The use of Mode 125, wherein the cells are delivered from the injection attachment under controlled pressure.

[0347] Use according to any one of embodiments 126 - 128, wherein the cells are not redistributed before loading into the injection accessory.

[0348] Use according to any one of embodiments 126 - 128, wherein the cells are redistributed before loading into the injection accessory.

[0349] Use according to embodiment 130, wherein the cells are redistributed by vortexing or manual agitation.

[0350] Use according to any one of embodiments 126 - 131, wherein the cells are subretinally injected.

[0351] Use according to any one of embodiments 126 - 132, wherein the cells are further defined as cell aggregates.

[0352] Use according to any one of embodiments 126 - 133, wherein the cells are further defined as single cells.

[0353] Use according to any one of embodiments 126 - 134, wherein the cells are in a formulation buffer.

[0354] Use according to embodiment 135, wherein the formulation buffer is a balanced salt solution.

[0355] Use according to embodiment 136, wherein the balanced salt solution further comprises benzonase and / or human serum albumin.

[0356] Use according to any one of embodiments 133 - 137, wherein the cell aggregates are not suspended when injected into the tissue of the subject.

[0357] Use according to any one of embodiments 133 - 138, wherein the cells are present in the cannula for at least 5 minutes between loading and injection to allow the aggregates to settle in the cannula hub.

[0358] Use according to any one of embodiments 133 - 138, wherein between loading and injection, the cells are present in the cannula for at least 5 minutes to allow the cells to settle in the cannula hub.

[0359] Use according to any one of embodiments 133 - 140, wherein the cannula points downward to allow the cells to settle in the cannula hub.

[0360] Use according to any one of embodiments 133 - 139, wherein the cell aggregates are photoreceptor precursor cell aggregates.

[0361] Use according to any one of embodiments 143.126 - 138, wherein the cells are retinal progenitor cells and / or photoreceptor precursor cells.

[0362] Use according to any one of embodiments 144.133 - 138, wherein the cell aggregates are retinal progenitor cells and / or photoreceptor precursor cells.

[0363] Use according to any one of embodiments 145.126 - 142, wherein the cells are injected in a volume of less than 200 μL.

[0364] Use according to any one of embodiments 146.126 - 145, wherein the cells are injected in a volume of less than 100 μL.

[0365] Use according to any one of embodiments 147.126 - 146, wherein the cells are injected in a volume of approximately 50 μL.

[0366] Use according to any one of embodiments 148.126 - 147, wherein at least 1 million cells are injected.

[0367] Use according to any one of embodiments 149.126 - 148, wherein at least 2 million cells are injected.

[0368] Use according to any one of embodiments 150.126 - 149, wherein at least 25% of the cells loaded into the injection attachment are injected into the eye of the subject.

[0369] Use according to any one of embodiments 151.126 - 150, wherein the eye disorder is a hereditary retinal disease, age - related macular degeneration (AMD), hereditary macular degeneration, Stargardt 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.

[0370] A kit comprising the device and cells according to any one of embodiments 101 - 123.

[0371] A method for preparing a cell composition for delivery, comprising:

[0372] (a) Concentrating the cell composition;

[0373] (b) Preparing a bulk dose of the cell composition;

[0374] (c) Aliquoting a master dose from the bulk dose; and

[0375] (d) Dispensing the aliquoted doses.

[0376] Method 154. The method of Method 153, wherein the cell composition is a cryopreserved cell composition.

[0377] Method 155. The method of Method 153, the method further comprising thawing and resuspending the cryopreserved cell composition before step (a).

[0378] Method 156. The method of any one of Methods 153-155, the method further comprising dissociating and counting the cells in the cell composition after preparing the main dose.

[0379] Method 157. The method of any one of Methods 153-156, wherein step (c) comprises adding a formulation buffer to reach a target concentration.

[0380] Method 158. The method of any one of Methods 153-157, wherein the main dose is 75 μL.

[0381] Method 159. The method of any one of Methods 153-158, wherein the aliquot dose is 50 μL.

[0382] Method 160. The method of any one of Methods 153-159, wherein the cell composition is resuspended in a formulation buffer.

[0383] Method 161. The method of any one of Method 160, wherein the formulation buffer comprises a balanced salt solution.

[0384] Method 162. The method of Method 160 or 161, wherein the formulation buffer further comprises albumin.

[0385] Method 163. The method of Method 162, wherein the albumin is human serum albumin.

[0386] Method 164. The method of any one of Methods 153-163, the method further comprising loading the cell composition into a device of any one of Methods 96-123.

[0387] Method 165. The method of any one of Methods 153-163, the method further comprising delivering the cell composition according to the method of any one of Methods 1-95.

[0388] ****

[0389] According to the present disclosure, all of the methods disclosed and claimed herein can be made and executed without undue experimentation. 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 changes can be made in the methods and the steps or the order 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 achieving the same or similar results. All such similar substitutions and modifications that are obvious 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.

[0390] References

[0391] The following references are specifically incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement those described herein.

[0392] U.S. Patent Publication No. 2002 / 0076747

[0393] International Patent Publication No. WO 98 / 30679

[0394] Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006

[0395] Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd Edition Cold Spring Harbor Lab. Press, 2001.

Claims

1. A method for cell transplantation using an injection accessory, the injection accessory 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 accessory such that the cannula points downward until the plurality of cells settle together near the distal end; Inserting the cannula into the tissue of a subject; And Injecting the plurality of cells into the tissue of the subject using the injection accessory.

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

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 at the distal end is smaller 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 with the cannula.

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

6. The method of claim 1, wherein the injection accessory is oriented such that the cannula points downward for at least 2 minutes.

7. The method of claim 1, wherein the injection accessory is oriented such that the cannula points downward for at least 5 minutes.

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

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

10. The method of claim 1, the method further comprising: Providing a plurality of cells to the injection accessory such that the hub is pre-filled.

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

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

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

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-ended.

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. A method for cell transplantation in an eye using an injection accessory, the injection accessory 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 accessory such that the cannula points downward until the plurality of cells settle together near the distal end; Inserting the cannula into the retinal tissue of a subject; And Injecting the plurality of cells into the retinal tissue of the subject using the injection accessory, wherein the injection accessory is oriented such that the cannula points downward for at least 2 minutes, wherein the hub includes a proximal end, a distal end, and an inner surface extending between the proximal end and the distal end, wherein the inner surface tapers inwardly between the proximal end and the distal end.

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

20. An injection accessory for cell transplantation in an eye, comprising: a hub having a proximal end, a distal end, and an inner surface extending therebetween, the inner surface defining an internal volume; and a cannula coupled to the distal end of the hub and in fluid communication with the internal volume, wherein the internal volume contains a pre-filled volume of cells, wherein the internal volume is generally funnel-shaped, wherein the inner surface of the hub does not overlap with the cannula, and wherein at least 90% of the cells settle near the distal end of the hub when the distal end of the hub points downward.

21. The injection accessory of claim 20, wherein 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.

22. The injection accessory of claim 200, wherein the inner surface tapers inwardly from the proximal end to the distal end such that the inner surface diameter at the distal end is less than the inner surface diameter at the proximal end, and wherein the internal volume does not retain cells after injection.

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