Methods for enhanced propagation of cells
Inventors
Szczypka, Mark • Tziampazis, Evangelos • Westover, Angela J.
Assignees
RENAMED BIOLOGICS Inc • INNOVATIVE BIO THERAPIES Inc • Seastar Medical Inc
Publication Number
US-9029144-B2
Publication Date
2015-05-12
Expiration Date
2029-06-16
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
The present invention relates generally to methods for the isolation and propagation of cells. For example, embodiments of the present invention relate to isolation and propagation methods for the manufacture of a large number of cells for use, for example, in biotherapeutic devices, such as devices for renal replacement therapy for the treatment of acute renal failure (ARF), acute tubular necrosis (ATN), multi-organ failure (MOF), sepsis, cardiorenal syndrome (CRS) and end-stage renal disease (ESRD).
Core Innovation
The invention relates to methods for enhanced propagation of cells, particularly renal cells, from a sample such as a kidney. The main objective is to efficiently isolate and expand precursor cell populations, resulting in significantly increased cell yields suitable for the manufacture of large numbers of cells for biotherapeutic devices. The methods are designed for clinical applications requiring substantial cell quantities, such as for devices used in renal replacement therapy to treat conditions including acute renal failure (ARF), acute tubular necrosis (ATN), multi-organ failure (MOF), sepsis, cardiorenal syndrome (CRS), and end-stage renal disease (ESRD).
The invention addresses the limitations of existing cell therapy methods, which yield insufficient quantities of renal epithelial cells (REC) for chronic or large-scale therapeutic needs. Current therapies often involve inefficient cell isolation processes that prioritize mature, differentiated cells, sacrificing the proliferation potential of precursors, and do not enable robust cell expansion needed to meet clinical demand. This gap is especially critical given the suboptimal outcomes and supply constraints in organ transplantation and dialysis, as well as the need for improved metabolic and endocrine support in renal failure patients.
To solve these challenges, the invention provides a protocol involving optimized enzymatic digestion to generate small tissue fragments (about 212 μm or less), the use of higher centrifugal forces (greater than about 50×(g) or 200×(g)), plating at low cell densities, retaining non-adherent cells during culture and subculture, and maintaining precursor cells in an undifferentiated state until just prior to use. The methods allow for repeated subculture at subconfluent densities and can optionally include the addition of retinoic acid for subsequent terminal differentiation. These improvements dramatically increase cell yields from limited starting material while ensuring that the propagated cells retain therapeutic potential and functional characteristics.
Claims Coverage
The patent includes three main independent claims, each directed to enhanced methods for propagating renal or other cells from kidney samples, with emphasis on precursor cell expansion, cell handling, and specific culture procedures.
Culturing renal precursor cells while retaining non-adherent cells during medium changes
This feature involves culturing renal precursor cells from a kidney sample and specifically retaining non-adherent cells whenever the culture or subculture growth medium is changed. Retention of non-adherent cells is performed throughout the process, thereby enhancing the total propagation and yield of the cell population from the sample.
Propagation of renal cells using fragmenting, centrifugation, and culture without retinoic acid
This method comprises: 1. Treating a kidney sample to produce tissue fragments of about 212 μm or less in size; 2. Harvesting fragments by centrifugation at greater than 200×(g) to produce a pelleted slurry; 3. Applying a portion of the slurry to a solid support and culturing the cells in the absence of retinoic acid to allow them to attach and divide; 4. Harvesting the cells prior to confluence; 5. Applying the harvested cells to a solid support and subculturing them, again in the absence of retinoic acid, to permit further division; 6. Finally, harvesting the propagated population of renal cells.
Enhanced propagation protocol with fragmenting, pelleted slurry plating, and low-density subculturing
This aspect includes: - Separating a kidney sample into fragments of about 212 μm or less to create a slurry; - Centrifuging the slurry at greater than 200×(g) to obtain a pelleted slurry; - Creating a culture by applying about 10 μL or less of the pelleted slurry per square centimeter of growth surface, with resuspension in culture medium as needed for plating; - Subculturing about 8.7×10^4 or fewer cells from the culture per square centimeter of subculture surface. This method emphasizes low-density plating and subculturing to maximize cell expansion.
The inventive features collectively cover methods for isolating and propagating renal precursor cells using improved handling of tissue fragments, higher centrifugation forces, retention of non-adherent cells, low plating and passage densities, and maintenance of undifferentiated states during propagation to achieve high-yield, therapeutically relevant cell populations.
Stated Advantages
The invention enables the propagation of significantly greater numbers of renal cells compared to historic methods, meeting clinical demand for cell-based therapeutic devices.
Enhanced propagation protocols improve manufacturing feasibility by increasing cell yield per donor kidney, translating into higher numbers of therapeutic devices that can be produced.
The methods retain the therapeutic potential and functional characteristics of the propagated cells, such as cytokine response and metabolic activity.
The invention allows for the use of small tissue samples, including biopsies, making autologous device manufacture possible and practical for chronic care applications.
Cryopreservation of precursor cells is feasible without loss of identity or viability, enabling manufacturing flexibility and continuity despite intermittent tissue availability.
Documented Applications
Manufacture of renal cell-based therapeutic devices for renal replacement therapy to treat acute renal failure (ARF), acute tubular necrosis (ATN), multi-organ failure (MOF), sepsis, cardiorenal syndrome (CRS), and end-stage renal disease (ESRD).
Use of enhanced propagation methods for preparing cells for hollow fiber-based renal assist devices (RAD) and wearable bioartificial kidney (WEBAK) devices.
Production of bioartificial renal epithelial cell systems (BRECS), including integration of propagated cells into carbon disk-based devices and their application in extracorporeal or peritoneal dialysis circuits.
Facilitation of autologous or allogenic cell collection, expansion, and use in biotherapeutic devices, including from biopsies for individualized therapy.
Interested in licensing this patent?