Method for agglutinating cells in suspension

Abstract

The invention features a method of producing aggregates of multiple cell populations (e.g., a first population of cells and a second population of cells). The method includes agitating a liquid medium containing the cells in a bioreactor. These aggregates may be used for subsequent downstream applications, such as encapsulation into a biocompatible scaffold to form an artificial tissue construct.

Description

[Technical field]

[0001] The present invention relates to a method for agglutinating cells in suspension. [Background technology]

[0002] Many diseases result from the injury, dysfunction, or loss of a single organ or tissue type. While certain strategies such as organ transplantation can be effective, there is a great demand for organ replacement. Tissue therapy, including the development of artificial tissue constructs (e.g., cell-based transplants), is one of the most promising multidisciplinary approaches to meet this demand. However, despite great advances in the fields of cell biology, microfluidics, and engineering, traditional approaches are currently unable to recapitulate functional tissues at the scale required to confer therapeutic efficacy. The formation of cell aggregates to generate tissues is important as a first step to create useful tissues. However, it remains difficult to mimic biological conditions to achieve such conditions. Thus, new methods to form cell aggregates are needed. Summary of the Invention

[0003] In one aspect, the invention features a method of producing aggregates of a plurality of cell populations, the aggregates comprising a first population of cells and a second population of cells. The method includes (a) agitating a liquid medium containing the first population of cells and the second population of cells in a bioreactor for a duration sufficient to form aggregates comprising the first population of cells and the second population of cells. The first population of cells and the second population of cells are in suspension within the liquid medium during the agitating step. The method produces aggregates of the first population of cells and the second population of cells. The method may further include (b) collecting the aggregates comprising the first population of cells and the second population of cells.

[0004] In some embodiments, the first population of cells includes stromal cells (e.g., fibroblasts). The fibroblasts may be, for example, primary fibroblasts, induced pluripotent stem cell (iPSC)-derived fibroblasts, or embryonic stem cell (ESC)-derived fibroblasts. In some embodiments, the fibroblasts are genetically engineered fibroblasts.

[0005] In some embodiments, the second population of cells includes parenchymal cells. The parenchymal cells may be, for example, hepatocytes (e.g., human hepatocytes, e.g., primary human hepatocytes) or hepatocyte progenitor cells. The hepatocytes may be, for example, primary hepatocytes (e.g., primary human hepatocytes), iPSC-derived hepatocytes, or ESC-derived hepatocytes. The hepatocytes may be genetically engineered hepatocytes. The parenchymal cells may include pancreatic cells (e.g., alpha cells, beta cells, gamma cells, delta cells, or epsilon cells, or combinations thereof) or pancreatic progenitor cells. The pancreatic cells may be, for example, primary human pancreatic cells, iPSC-derived pancreatic cells, or ESC-derived pancreatic cells. In some embodiments, the pancreatic cells are genetically engineered pancreatic cells.

[0006] In some embodiments, the first population of cells comprises fibroblasts and the second population of cells comprises hepatocytes. In some embodiments, the first population of cells comprises fibroblasts and the second population comprises hepatocyte progenitor cells.

[0007] In some embodiments, the first population of cells comprises fibroblasts and the second population of cells comprises pancreatic beta cells. In some embodiments, one of the populations of cells comprises endocrine cells, exocrine cells, paracrine cells, heterocrine cells, autocrine cells, or juxtacrine cells.

[0008] In some embodiments, one of the populations of cells comprises Leydig cells, adrenal cortical cells, pituitary cells, thyroid cells, granulosa cells, mammary epithelial cells, thymic cells, thymic epithelial cells, hypothalamic cells, skeletal muscle cells, smooth muscle cells, and / or neuronal cells.

[0009] In some embodiments, the pituitary cells comprise thyrotropin-producing pituitary cells, lactotropin-producing pituitary cells, adrenocorticotropin-producing pituitary cells, growth hormone-producing pituitary cells, and / or gonadotropin-producing pituitary cells.

[0010] In some embodiments, the neuronal cells comprise dopaminergic cells. In some embodiments, one of the populations of cells comprises parenchymal cells (e.g., hepatocytes, pancreatic exocrine cells, muscle cells, pancreatic endocrine cells, neurons, intestinal cells, adipocytes, spleen cells, kidney cells, bile duct cells, Kupffer cells, stellate cells, cardiomyocytes, alveolar epithelial cells, bronchiolar cells, club cells, urothelial cells, mucus cells, parietal cells, chief cells, G cells, goblet cells, enteroendocrine cells, Paneth cells, M cells, tuft cells, glial cells, gallbladder cells, keratinocytes, melanocytes, Merkel cells, Langerhans cells, osteocytes, osteoclasts, esophageal cells, photoreceptor cells, and corneal epithelial cells). In some embodiments, the parenchymal cells are pancreatic cells (e.g., alpha cells, beta cells, gamma cells, delta cells, epsilon cells, or any combination thereof). In some embodiments, the parenchymal cells comprise beta cells.

[0011] In some embodiments, the cells are engineered cells, primary cells, or transdifferentiated cells. In some embodiments, the ratio of the first population of cells (e.g., stromal cells, e.g., fibroblasts) to the second population of cells (e.g., parenchymal cells, e.g., hepatocytes, e.g., primary human hepatocytes) is 10:1 to 1:10, 5:1 to 1:5, 3:1 to 1:3, or 2:1 to 1:2 (e.g., 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 1:10 to 1:1 to 1:2, 1:1 to 1:3, 1:1 to 1:4, 1:1 to 1:5, 1:1 to 1:6, 1:1 to 1:7, 1:1 to 1:8, 1:1 to 1:9, or 1:1 to 1:10). For example, the ratio of the first population of cells to the second population of cells can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the ratio is 2:1.

[0012] In some embodiments, the first population of cells is greater than or equal to 1×10 4 cells / mL~1×10 8 The first population of cells is present at a density of 1 x 10 cells / mL. For example, the first population of cells is present at a density of 1 x 10 4 cells / mL~1×10 5 cells / mL (e.g., 1 x 10 4 cells / mL, 2×10 4 cells / mL, 3×10 4 cells / mL, 4×10 4 cells / mL, 5×10 4 cells / mL, 6×10 4 cells / mL, 7×10 4 cells / mL, 8×10 4 cells / mL, 9×10 4 cells / mL, or 1 x 10 5 cells / mL), 1×10 5 cells / mL~1×10 6 cells / mL (e.g., 1 x 10 5 cells / mL, 2×10 5 cells / mL, 3×10 5 cells / mL, 4×10 5 cells / mL, 5×10 5cells / mL, 6×10 5 cells / mL, 7×10 5 cells / mL, 8×10 5 cells / mL, 9×10 5 cells / mL, or 1 x 10 6 cells / mL), 1×10 6 cells / mL~1×10 7 cells / mL (e.g., 1 x 10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, or 1 x 10 7 cells / mL), or 1 × 10 7 cells / mL~1×10 8 cells / mL (e.g., 1 x 10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, 4×10 7 cells / mL, 5×10 7 cells / mL, 6×10 7 cells / mL, 7×10 7 cells / mL, 8×10 7 cells / mL, 9×10 7 cells / mL, or 1 x 10 8 The cells may be present at a density of 1000 x 1000 cells / mL.

[0013] In some embodiments, the first population of cells comprises fibroblasts (e.g., primary fibroblasts, e.g., primary human fibroblasts), and the fibroblasts have a density of 1×10 5 cells / mL~1×10 7 cells / mL (e.g., 1 x 10 5 cells / mL, 2×10 5 cells / mL, 3×10 5 cells / mL, 4×10 5 cells / mL, 5×10 5 cells / mL, 6×10 5cells / mL, 7×10 5 cells / mL, 8×10 5 cells / mL, 9×10 5 cells / mL, 1×10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, or 1 x 10 7 For example, the density of fibroblasts is, for example, 6 × 10 5 It may be in cells / mL.

[0014] In some embodiments, the second population of cells is 1×10 4 cells / mL~1×10 8 For example, the second population of cells is present at a density of 1 x 10 4 cells / mL~1×10 5 cells / mL (e.g., 1 x 10 4 cells / mL, 2×10 4 cells / mL, 3×10 4 cells / mL, 4×10 4 cells / mL, 5×10 4 cells / mL, 6×10 4 cells / mL, 7×10 4 cells / mL, 8×10 4 cells / mL, 9×10 4 cells / mL, or 1 x 10 5 cells / mL), 1×10 5 cells / mL~1×10 6 cells / mL (e.g., 1 x 10 5 cells / mL, 2×10 5 cells / mL, 3×10 5 cells / mL, 4×10 5 cells / mL, 5×10 5 cells / mL, 6×10 5 cells / mL, 7×10 5 cells / mL, 8×10 5cells / mL, 9×10 5 cells / mL, or 1 x 10 6 cells / mL), 1×10 6 cells / mL~1×10 7 cells / mL (e.g., 1 x 10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, or 1 x 10 7 cells / mL), or 1 × 10 7 cells / mL~1×10 8 cells / mL (e.g., 1 x 10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, 4×10 7 cells / mL, 5×10 7 cells / mL, 6×10 7 cells / mL, 7×10 7 cells / mL, 8×10 7 cells / mL, 9×10 7 cells / mL, or 1 x 10 8 They may be present at a density of cells / mL.

[0015] In some embodiments, the second population of cells comprises hepatocytes (e.g., primary hepatocytes, e.g., primary human hepatocytes), and the density of the hepatocytes is 1×10 5 cells / mL~1×10 7 cells / mL (e.g., 1 x 10 5 cells / mL, 2×10 5 cells / mL, 3×10 5 cells / mL, 4×10 5 cells / mL, 5×10 5 cells / mL, 6×10 5 cells / mL, 7×10 5 cells / mL, 8×10 5 cells / mL, 9×10 5 cells / mL, 1×106 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, or 1 x 10 7 For example, the density of hepatocytes is, for example, 3 × 10 5 It may be in cells / mL.

[0016] In some embodiments, the plurality of cell populations further comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional cell populations. In some embodiments, the bioreactor has a volume of 0.1 L to 500 L, e.g., 0.1 L to 10 L (0.1 L, 0.2 L, 0.3 L, 0.4 L, 0.5 L, 0.6 L, 0.7 L, 0.8 L, 0.9 L, or 1 L), 1 L to 10 L (e.g., 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, or 10 L), 10 L to 100 L (e.g., 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, or 100 L), or 100 L to 500 L (e.g., 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 450 L, or 500 L). In some embodiments, the volume of the bioreactor is between 100 L and 300 L. In some embodiments, the volume of the bioreactor is between 0.1 L and 1 L (e.g., 0.5 L).

[0017] In some embodiments, the first population of cells and the second population of cells do not grow by more than 30% (e.g., in cell number) during the agitation step. For example, in some embodiments, the first population of cells and the second population of cells do not grow by more than 230%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0018] The bioreactor used in the methods described herein may be a stirred tank bioreactor (e.g., a disposable stirred tank bioreactor) or a vertical wheel bioreactor. The bioreactor may further comprise a bioprocessing controller to control one or more of pH, temperature, and dissolved oxygen concentration.

[0019] In some embodiments, the first population of cells and the second population of cells do not adhere to the bioreactor. In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an average diameter within ±10% (e.g., ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of each other.

[0020] In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an average diameter of less than 200 μm. In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an area / perimeter ratio of 45 or less, e.g., 40, 35, 30, 25, 20, 15, or 10 or less.

[0021] In some embodiments, the aggregates are spheroids. In some embodiments, the stirring step has a duration of 1 hour to 72 hours (e.g., 6 hours to 72 hours, 6 hours to 48 hours, 12 hours to 48 hours, or 12 hours to 24 hours). For example, in some embodiments, the stirring step comprises a duration of up to 48 hours. In some embodiments, the stirring step comprises a duration of up to 24 hours. In some embodiments, the stirring step comprises a duration of up to 18 hours. In some embodiments, the stirring step comprises a duration of up to 12 hours. In some embodiments, the stirring step comprises a duration of 12 to 24 hours. In some embodiments, the stirring step comprises a duration of 12 to 48 hours. In some embodiments, the stirring step comprises a duration of 24 to 48 hours.

[0022] In some embodiments, agitation comprises rotating the bioreactor at a speed of between 10 revolutions per minute (RPM) and 50 RPM (e.g., 15 RPM, 20 RPM, 25 RPM, 30 RPM, 35 RPM, 40 RPM, 45 RPM, or 50 RPM, e.g., 36 RPM).

[0023] In some embodiments, the viscosity of the medium is between 0.5 cP and 2 cP (e.g., between 0.9 cP and 1.4 cP, e.g., 0.5 cP, 0.6 cP, 0.7 cP, 0.8 cP, 0.9 cP, 1 cP, 1.1 cP, 1.2 cP, 1.3 cP, 1.4 cP, 1.5 cP, 1.6 cP, 1.7 cP, 1.8 cP, 1.9 cP, or 2 cP).

[0024] In some embodiments, the medium contains 1-20 μg / mL (e.g., 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, or 20 μg / mL) recombinant human insulin, 1-10 μg / mL (e.g., 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, or 10 μg / mL) human transferrin, and 1×10 -3 ~1×10 -2 μg / mL (e.g., 1 × 10 -3 μg / mL, 2×10 -3 μg / mL, 3×10 -3 μg / mL, 4×10 -3 μg / mL, 5×10 -3 μg / mL, 6×10 -3 μg / mL, 7×10 -3 μg / mL, 8×10 -3 μg / mL, 9×10 -3 μg / mL, or 1×10 -2 μg / mL) of selenite.

[0025] In some embodiments, the medium contains laminin, collagen, elastin, or fibronectin. For example, the medium may contain 1 to 10 μg / mL (e.g., 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, or 10 μg / mL) of laminin. In some embodiments, the medium contains fibrinogen.

[0026] In some embodiments, the medium contains 1 to 20 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM) of a Rho-associated protein kinase (ROCK) inhibitor. The ROCK inhibitor may be, for example, Y27632.

[0027] In some embodiments, the medium comprises human serum. For example, the medium may comprise 0.1% to 20% (v / v) human serum, for example, 0.1% to 1% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), for example, 1% to 10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%), or 10% to 20% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some embodiments, the medium comprises 1% to 10% (v / v) human serum. In some embodiments, the medium comprises 10% (v / v) human serum.

[0028] In some embodiments, the medium comprises a platelet lysate (e.g., human platelet lysate, e.g., PLATELET GOLD™). For example, the medium may comprise 0.1%-10% (v / v) platelet lysate, e.g., 0.1%-1% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 1%-10% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) human platelet lysate. In some embodiments, the medium comprises 1%-5% (v / v) platelet lysate.

[0029] In some embodiments, the medium comprises glucose (e.g., 4,000 mg / L to 5,000 mg / L glucose, e.g., 4,100 mg / L, 4,200 mg / L, 4,300 mg / L, 4,400 mg / L, 4,500 mg / L, 4,600 mg / L, 4,700 mg / L, 4,800 mg / L, 4,900 mg / L, or 5,000 mg / L glucose). In some embodiments, the medium comprises 4,500 mg / L glucose.

[0030] In some embodiments, the medium comprises glucagon (e.g., 10 ng / mL to 100 ng / mL of glucagon, e.g., 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL of glucagon). In some embodiments, the medium comprises 40 ng / mL of glucagon.

[0031] In some embodiments, the medium comprises dexamethasone (e.g., 10 ng / mL to 100 ng / mL dexamethasone, e.g., 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL dexamethasone). In some embodiments, the medium comprises 60 ng / mL dexamethasone.

[0032] In some embodiments, the bioreactor is incubated at a temperature between 35° C. and 39° C. (e.g., 35° C., 35.5° C., 36° C., 36.5° C., 37° C., 37.5° C., 38° C., 38.5° C., or 39° C.).

[0033] In some embodiments, the method produces aggregate densities of 500 aggregates / mL to 10,000 aggregates / mL (e.g., 500 aggregates / mL, 600 aggregates / mL, 700 aggregates / mL, 800 aggregates / mL, 900 aggregates / mL, 1,000 aggregates / mL, 2,000 aggregates / mL, 3,000 aggregates / mL, 4,000 aggregates / mL, 5,000 aggregates / mL, 6,000 aggregates / mL, 7,000 aggregates / mL, 8,000 aggregates / mL, 9,000 aggregates / mL, or 10,000 aggregates / mL).

[0034] In some embodiments, the method produces an average aggregate mean diameter of 50 μm to 200 μm (e.g., 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, or 200 μm).

[0035] In some embodiments, the method produces an average total volume of aggregates between 200 μL and 50 mL, e.g., between 200 μL and 1 mL (e.g., 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 550 μL, 600 μL, 650 μL, 700 μL, 750 μL, 800 μL, 850 μL, 900 μL, 950 μL, or 1 mL), between 1 mL and 10 mL (e.g., 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL), or between 10 mL and 100 mL (e.g., 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL).

[0036] In some embodiments, the method produces an average total mass of aggregates of between 100 mg and 100 g, e.g., between 100 mg and 1 g (e.g., 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1 g), between 1 g and 10 g (e.g., 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, or 10 g), or between 10 g and 100 g (e.g., 10 g, 20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, or 100 g).

[0037] In some embodiments, collecting the aggregates comprises pipetting, pouring, decanting, or draining the bioreactor. In some embodiments, the bioreactor comprises a collection tube and the method comprises collecting the aggregates in the collection tube.

[0038] In some embodiments, the method further comprises washing the collected aggregates of step (b). In some embodiments, the method further comprises purifying the collected aggregates of step (b). The collected aggregates may be purified, for example, by centrifugation or acoustic separation. For example, the collected aggregates may be purified by countercurrent centrifugation or density gradient centrifugation.

[0039] In some embodiments, at least 80% (e.g., at least 80%, 85%, 90%, 95%, 97%, or 99%) of the aggregates have an average diameter of each other after purification within ±10% (e.g., ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0040] In some embodiments, the method further comprises concentrating the collected aggregates of step (b). In some embodiments, the method further comprises formulating the collected aggregates of step (b) in a storage buffer.

[0041] In some embodiments, the biocompatible scaffold further comprises a reinforcing agent, which may include, for example, collagen, poly(ethylene glycol), polyvinylidene acetate (PVDA), polyvinylidene fluoride (PVDF), poly(lactic-co-glycolic acid) (PLGA), or poly(L-lactic acid) (PLLA).

[0042] In some embodiments, each of the aggregates (e.g., at least 80%, e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates has a molecular weight of at least 1 × 10 3 cells (e.g., at least 1 x 10 3 cells, 2 x 10 3 cells, 3 x 10 3 cells, 4 x 10 3 cells, 5 x 10 3 cells, 6 x 10 3 cells, 7 x 10 3 cells, 8 x 10 3 cells, 9 x 10 3 cells, or 1 x 10 4 In some embodiments, the aggregates each contain at least 1 x 10 5 cells, 1 x 10 6 cells, 1 x 10 7 cells, or 1 x 10 8 Contains cells.

[0043] In some embodiments, the aggregates (e.g., at least 80% of the aggregates, e.g., at least 85%, 90%, 95%, 97%, or 99%) each have an average diameter of at least 50 μm (e.g., at least 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, or more).

[0044] In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an average diameter of less than 200 μm. In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an area / perimeter ratio of 45 or less, e.g., 40, 35, 30, 25, 20, 15, or 10 or less.

[0045] In some embodiments, the method further comprises washing the cells prior to step (a), for example after thawing the cells. In some embodiments, the method further comprises (c) encapsulating the collected aggregates of step (b) within a biocompatible scaffold.

[0046] In another aspect, the invention features a method of encapsulating aggregates of multiple cell populations, including a first population of cells and a second population of cells. The method includes (a) agitating a liquid medium containing the first population of cells and the second population of cells in a bioreactor for a duration sufficient to form aggregates including the first population of cells and the second population of cells. The first population of cells and the second population of cells are in suspension in the liquid medium during the agitating step. The method produces aggregates of the first population of cells and the second population of cells. The method further includes (b) collecting the aggregates of step (a), e.g., aggregates including the first population of cells and the second population of cells. The method also includes (c) encapsulating the aggregates of step (b) in a biocompatible scaffold.

[0047] In some embodiments, encapsulating the aggregates includes providing a polymerization agent or cross-linking reagent to polymerize or cross-link the biocompatible scaffold, thereby encapsulating the aggregates.

[0048] The biocompatible scaffold may include, for example, fibrinogen. The polymerization agent may include, for example, thrombin. In some embodiments, thrombin polymerizes fibrinogen into fibrin.

[0049] In some embodiments, the method further comprises washing the collected aggregates of step (b) prior to encapsulation. In some embodiments, the method further comprises washing the cells prior to step (a), for example after thawing the cells.

[0050] In some embodiments, the method further comprises purifying the collected aggregates of step (b) prior to encapsulation. The collected aggregates may be purified, for example, by centrifugation or acoustic separation. For example, the collected aggregates may be purified by countercurrent centrifugation or density gradient centrifugation.

[0051] In some embodiments, at least 80% (e.g., at least 80%, 85%, 90%, 95%, 97%, or 99%) of the aggregates have an average diameter of each other after purification within ±10% (e.g., ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0052] In some embodiments, the method further comprises concentrating the collected aggregates of step (b) prior to encapsulation. In some embodiments, the method further comprises formulating the collected aggregates of step (b) in a storage buffer prior to encapsulation.

[0053] definition The term "agitation" as used herein refers to moving the liquid medium through the cell culture so that the contents of the cell culture remain in suspension and do not settle or adhere to the bioreactor. Agitation can be accomplished by any suitable method known in the art, such as, for example, stirring, shaking, rotating, spinning at high speed, etc.

[0054] An "artificial tissue construct" as used in the context of this disclosure refers to a mixture of cultured cells (e.g., parenchymal cells (e.g., hepatocytes (e.g., primary human hepatocytes)) and, optionally, stromal cells (e.g., fibroblasts, e.g., neonatal foreskin fibroblasts) and a biocompatible scaffold (e.g., a biocompatible hydrogel scaffold, e.g., fibrin). The relative volume of the artificial tissue construct may range from 0.1 mL to 5 L.

[0055] The cells may be from an established cell line or may refer to primary cells, and herein "primary cells", "primary cell lines", and "primary cultures" are used interchangeably to refer to cells and cell cultures that are derived from a limited number of passages (e.g., splits) of culture and can be expanded in vitro. For example, a primary culture may refer to a culture that has been passaged 0, 1, 2, 4, 5, 10, or 15 times, but has not been passaged enough times to pass through a crisis phase. A primary cell line can be maintained in vitro for less than 10 passages. When the cells are primary cells, such cells can be harvested from an individual in any convenient manner. For example, cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be conveniently harvested by biopsy. A suitable solution can be used to disperse or suspend the harvested cells. Such solutions are generally balanced salt solutions such as saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc., generally at low concentrations of 5-25 mM, along with acceptable buffers, supplemented as appropriate with fetal bovine serum or other naturally occurring factors. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. The cells can be used immediately or frozen and stored for long periods of time and thawed for reuse. In such cases, the cells are typically frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution commonly used in the art to store the cells at such freezing point temperatures, and thawed in a manner commonly known in the art for thawing frozen cultured cells. For example, hepatocytes may be isolated by conventional methods (Berry and Friend, 1969, J. Cell Biol. 43:506-520), which can be adapted to human liver biopsy or autopsy material (e.g., to obtain primary human hepatocytes).

[0056] As used herein, the term "cell type" refers to a group of cells that share a statistically separable phenotype based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of a common cell type may include cells isolated from a common tissue (e.g., epithelial, nervous, connective, or muscle tissue) and / or cells isolated from a common organ, tissue system, blood vessel, or other structure and / or region in the body.

[0057] As used herein, a scaffold (e.g., a hydrogel scaffold) is considered "biocompatible" if it does not exhibit toxicity when introduced into a subject (e.g., a human). In view of the present disclosure, a biocompatible scaffold preferably does not exhibit toxicity to cells of an artificial tissue construct or when implanted in vivo into a subject (e.g., a human). For example, with respect to hepatocytes, hepatotoxicity can be measured by, for example, determining hepatocyte apoptotic mortality (e.g., increased apoptosis is indicative of hepatotoxicity), aminotransferase levels (e.g., increased aminotransferase levels are indicative of hepatotoxicity), hepatocyte ballooning (e.g., increased ballooning is indicative of hepatotoxicity), hepatocyte droplet steatosis (e.g., increased steatosis is indicative of hepatotoxicity), cholangiocyte mortality (e.g., increased cholangiocyte mortality is indicative of hepatotoxicity), and gamma-glutamyl transpeptidase (GGT) levels (e.g., increased GGT levels are indicative of hepatotoxicity). Biocompatible scaffolds can include, but are not limited to, fibrin and heparin. The biocompatible scaffold may be a biocompatible hydrogel scaffold.

[0058] The term "hydrogel" as used herein refers to a network of polymer chains that is hydrophilic in nature such that the material absorbs large amounts of water or other aqueous solutions. Hydrogels can contain, for example, at least 70% v / v water, at least 80% v / v water, at least 90% v / v water, at least 95%, 96%, 97%, 98%, or even 99% or more v / v water (or other aqueous solutions). Hydrogels may contain natural or synthetic polymers, and the polymer network is often characterized by a high degree of cross-linking. Because of their high water content, hydrogels also have a degree of flexibility very similar to natural tissues. Hydrogels are particularly useful in tissue engineering applications as scaffolds for culturing cells. In certain embodiments, hydrogels are made from biocompatible polymers. [Brief description of the drawings]

[0059] [Figure 1A] (A and B) are graphs showing the number of cells per mL as a function of time when aggregated in suspension in ITS (insulin-transferrin-selenium) medium alone or with the addition of laminin or ROCK inhibitor. A shows live cells and B shows dead cells. [Figure 1B] Same as above. [Figure 2A] Graphs A to D show aggregation data under various culture conditions. A shows the number of seeds (i.e., aggregates). B shows the average diameter of aggregates. C shows the number of debris. D shows the number of superaggregates (SuperAgg). Note that the microwell baseline is a comparison condition in which cells aggregated in the microwells. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Diagram 3] 1 is a series of graphs showing aggregate (seed) diameter distribution in various culture conditions. [Figure 4] 1 is a set of photomicrographs showing superaggregates formed under different culture conditions. [Diagram 5] Graphs A-D show aggregation data for various culture conditions at 24 or 48 hours of culture. A shows seed / aggregate number. B shows average aggregate diameter. C shows debris number. D shows superaggregate number. [Figure 6] 1 is a set of photomicrographs showing aggregates formed under various culture conditions. [Figure 7] Graphs A-D show aggregate (seed) data with ITS alone or in the presence of ROCK inhibitor. A shows aggregate (seed) size in different replicates. B shows aggregate (seed) concentration in different replicates. C shows aggregate (seed) size after different replicates. D shows debris concentration after different replicates. [Figure 8] A and B are graphs showing ammonia clearance. A shows ammonia clearance. B shows the same data with the cell-free background subtracted. [Figure 9] Graphs showing ammonia clearance in aggregates formed under suspension conditions compared to aggregates formed in microwells (A) and (B) show ammonia clearance compared to aggregates formed in microwells at 18 and 42 hours. [Figure 10] 1 is a table showing aggregate (seed) properties under various culture conditions with ITS alone or in the presence of ROCK inhibitors. [Figure 11] 1 is an exemplary set of micrographs and graphs showing aggregate (seed) size for aggregates formed in an ITS: Seed number: 2052, Seed concentration (assuming 300 μL): 6840.0 seeds / mL, Seed average diameter: 86.8 μm, Seed median diameter: 81.6 μm, Debris score: 11321, Superaggregate number: 2. [Figure 12]1 is an exemplary set of micrographs and graphs showing aggregate (seed) size for aggregates formed in the presence of a ROCK inhibitor: seed number: 1160, seed concentration (assuming 300 μL): 3866.7 seeds / mL, seed mean diameter: 102.5 μm, seed median diameter: 97.3 μm, debris score: 2045, superaggregate number: 1. [Figure 13] Graphs A and B show the concentration of cells over 24 hours of culture in suspension conditions, A shows live cells and B shows dead cells. [Figure 14] Graphs A-D show aggregation data at various concentrations of human serum (HS) and platelet gold (PG; human lysate). A shows aggregate (seed) size. B shows average aggregate (seed) volume. C shows aggregate (seed) concentration. D shows total aggregate (seed) volume. [Figure 15] A-D are graphs showing aggregation data generated under various culture conditions. A shows aggregate (seed) size. B shows average aggregate (seed) volume. C shows average aggregate (seed) volume. D shows total aggregate (seed) volume. VWB_005 and VWB_006 are ITS, fibrinogen, ROCK inhibitor, laminin, and 10% fetal bovine serum. VWB_007 is human serum and platelet lysate. [Figure 16] 1 is a graph showing lactate dehydrogenase (LDH) formed after 24 hours under various culture conditions, including ITS, Platelet Gold and human serum conditions. [Figure 17] 13 is a set of photomicrographs showing live and dead fluorescent staining channels overlaid on a phase contrast image of aggregates harvested after 24 hours. [Figure 18] 1 is a graph showing the number of live and dead cells (normal human dermal fibroblasts) after thawing and washing with a CTS™ ROTEA™ harvest using a counterflow centrifugation system. Viability remained unchanged at 98% to 97%. Recovery was approximately 87%. [Figure 19]Graphs A-C show the number of live and dead cells (primary human hepatocytes (PHH)) after washing using the CTS™ ROTEA™ counterflow centrifugation system at A: 30 mL / min, B: 60 mL / min, and C: 17 mL / min. [Figure 20] Graphs A and B show viable cells after washing using a CTS™ ROTEA™ counterflow centrifugation system. A shows the percentage of viable cells as a function of flow rate. B shows the % change in dead cell count as a function of flow rate. [Figure 21] 1 is a set of graphs showing aggregate (seed) data before (left) and after (right) washing of microwell-based aggregates using a CTS™ ROTEA™ counter-flow centrifugation system. [Figure 22] 1 is a graph showing recovery as a function of aggregate size. [Diagram 23] 1 is a set of graphs showing aggregate (seed) data before (top) and after (bottom) vertical wheel bioreactor-based aggregate washing and concentration using a CTS™ ROTEA™ countercurrent centrifugation system. [Figure 24] Graph showing estimated percent yield as a function of aggregate size in two replicate experiments. [Diagram 25] A–D are a set of graphs showing seed number (A), seed concentration (B), seed diameter (C), and debris number (D) for the indicated conditions (ITS, 10% human serum, 5% human serum, or 5% Platelet Gold) after 24 h of aggregation. [Figure 26] Graph showing superaggregate numbers in the indicated media conditions after 24 hours of suspension-induced aggregation. [Figure 27] A and B are graphs showing average seed volume and seed volume per mL of culture for the indicated medium conditions. [Figure 28]A and B are graphs showing normalized values ​​of LDH (mU) per reactor (aggregation) (A) and per plate (encapsulation) (B). HS is human serum and PG is PLATELET GOLD™ human platelet lysate. [Figure 29] A and B are graphs showing alanine aminotransferase (ALT) (U / L) (A) and aspartate aminotransferase (AST) (U / L) (B) released by PHH and NHDF cell suspensions during 24 h of VWB aggregation. n=3 samples from each reactor condition. Means and 95% confidence intervals are plotted. Plotted values ​​represent initial levels of ALT and AST at 0 h (onset of aggregation). [Diagram 30] A and B are graphs showing ammonia clearance. A shows normalized ammonia concentrations including the no-cell control. B shows relative ammonia for experimental groups minus / removal of the no-cell control. n=4 for each condition, mean values ​​and 95% confidence intervals are plotted. The dashed line represents a relative ammonia concentration of 1.0 (100% relative ammonia concentration). [Diagram 31] FIG. 1 is a graph showing relative levels of ammonia removed after 72 hours. n=4 grafts per condition. Mean values ​​and 95% confidence intervals are plotted. [Diagram 32] FIG. 1 is a graph showing ammonia clearance k values ​​for grafts made from the various flocculation media formulations tested. n=4 grafts per condition, averages are plotted. Constraint: k>0. [Diagram 33] 1 is a graph showing seed concentration as a function of aggregation time for the indicated experimental conditions. [Diagram 34] 1 is a graph showing seed diameter as a function of aggregation time for the indicated experimental conditions. [Diagram 35] A and B are graphs showing the total number of remaining cells (A) or the total number of dead cells (B) as a function of aggregation time for the indicated experimental conditions. [Diagram 36]A and B are graphs showing AST secreted during aggregation as a function of aggregation time (A) and AST normalized to PHH concentration as a function of aggregation time (B) for the experimental conditions indicated. [Figure 37] Graph showing seed viability as a function of aggregation time for the indicated experimental conditions. [Figure 38] A and B are a set of graphs showing U1 potency screening of ammonia clearance in the presence of control (A) or subtracting control (B). [Figure 39] A and B are graphs showing U1-surroseed total signal (human albumin) in the presence of FIBRYGA® control (A) or U1-surroseed total signal (human albumin) when FIBRYGA® control is subtracted (B). Groups: 1: microwell, Lonza, RUO fibrin; 2: VWB, ABM, FIBRYGA®; 3: microwell, ABM, FIBRYGA®; 4: microwell, ABM, RUO fibrin; 5: VWB, ABM, RUO fibrin; 6: microwell, Lonza, FIBRYGA®; 7: GMP FIBRYGA® (cell-free control). [Diagram 40] FIG. 1 is a schematic showing the timeline of graft implantation in mice. [Diagram 41] A-C are graphs showing trends of in vivo plasma biomarkers. A is albumin, B is transferrin, and C shows the correlation between albumin and transferrin. Groups: 1: Baseline (Microwell, Expanded Lonza, RUO Fibrin); 2: Novel (VWB, ABM T&A, FIBRYGA®); 3: Control (Microwell, ABM, ABM T&A, GMP Fibrin); 4: Control (Microwell, ABM T&A, RUO Fibrin); 5: Control (VWB, ABM T&A, RUO Fibrin); 6: Control (cell-free GMP Fibrin). [Diagram 42] 1 is an image showing an example of a CK18 stained liver cell image. [Diagram 43] 1 is an image showing an example of a CD31 stained blood vessel image. [Diagram 44] 13 is an image showing an example of detection of hepatocyte aggregates by the cell profiler. [Diagram 45] 13 is an image showing an example of magnifying an area around hepatocytes to define a region of interest for vascular analysis. [Figure 46] 13 is an image showing an example of a mask resulting from ROI definition based on the location of liver cells. [Figure 47] 13 is an image showing an example of a CD31 vascular region selected by a mask. [Figure 48] Graph showing average vessel length. Data are means and 95% confidence intervals. [Figure 49] 1 is a graph showing the number of hepatocyte aggregates per mm2. [Figure 50] 1 is a graph showing the number of blood vessels per mm2. [Figure 51] 1 is a graph showing the number of blood vessels per unit number of hepatocyte aggregates. [Figure 52] FIG. 1 is a graph showing correlation assessment between secreted human albumin levels and aggregate density. [Diagram 53] FIG. 13 is a graph showing correlation assessment between blood vessel number per unit aggregate number and secreted albumin. [Figure 54] FIG. 13 is a graph showing seed bed height as a function of seed dose density produced by a vertical wheel bioreactor with and without washing by a ROTEA™ countercurrent centrifugation system. [Figure 55] 1 is a series of images showing graft cross sections for seed bed heights of various doses and seed types. VWB is a vertical wheel bioreactor. [Figure 56] A and B are graphs showing corrected ALT levels (A) and AST levels (B) in aggregate cell volumes of 60 mL, 75 mL, 100 mL, and 375 mL at 0 and 22 hours. [Figure 57]1 is a graph showing oxygen tension in aggregated cells of 60 mL, 75 mL, 100 mL, and 375 mL volumes over 22 hours. [Figure 58] Graphs showing lactate dehydrogenase (LDH) concentrations (mU) from normal human dermal fibroblasts (NHDF), primary human hepatocytes (PHH), and a combination of NHDF and PHH at 0, 24, and 60 hours (A and B). A shows extracellular LDH and B shows total LDH. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] The formation of cell aggregates for tissue engineering is important as a first step to create tissues useful for various therapeutic approaches. However, it remains difficult to mimic biological conditions to achieve such conditions. In particular, many methods utilize two-dimensional approaches, which may only create flat layers of tissue compared to the three-dimensional structures required for many tissues. Moreover, employing easy manipulation of cell aggregates remains challenging due to the need for exchange of various cell media to ensure robust cell growth and aggregation.

[0061] The present invention solves this problem by providing a bioreactor-based method for producing aggregates in suspension in a liquid medium. The method includes providing a plurality of cell populations, including a first population of cells and a second population of cells, and agitating the liquid medium containing the cell populations in a bioreactor for a duration sufficient to form aggregates of the first population of cells and the second population of cells. The present invention is based in part on the surprising discovery that aggregates of two distinct populations of cells can be formed in agitated liquid medium in a bioreactor, even though the cells are maintained in suspension and minimal cell-to-cell contact is assumed to be necessary for aggregation. Moreover, the method reduces, eliminates, or minimizes proliferation while promoting aggregation, thus creating cell aggregates useful for a variety of downstream applications, such as encapsulation in biocompatible scaffolds for tissue and organ engineering. Finally, the use of a suspension-based bioreactor allows for the scalability of producing commercially relevant quantities of cell aggregates.

[0062] Method for Producing Aggregates The invention features a method of producing aggregates of multiple cell populations (e.g., a first population of cells and a second population of cells). The method includes agitating a liquid medium containing the first population of cells and the second population of cells in a bioreactor. The first population of cells and the second population of cells are in suspension in the liquid medium during the agitating step. The cells are agitated for a duration sufficient to form aggregates comprising the first population of cells and the second population of cells. The method may further include collecting the aggregates of the first population of cells and the second population of cells. These aggregates may be used for subsequent downstream applications, such as encapsulation in a biocompatible scaffold to form an artificial tissue construct. Prior to encapsulation, the aggregates may be collected, washed, purified, concentrated, and / or formulated in a suitable storage medium (e.g., a buffer).

[0063] In some embodiments, the first population of cells and the second population of cells do not grow by more than 30% (e.g., in cell number) during the agitation step. For example, in some embodiments, the first population of cells and the second population of cells do not grow by more than 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0064] In some embodiments, the first population of cells and the second population of cells do not adhere to the bioreactor. In some embodiments, the stirring step is for 1 hour to 72 hours (e.g., 6 hours to 72 hours, 6 hours to 48 hours, 12 hours to 48 hours, or 12 hours to 24 hours, e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, or 72 hours). For example, in some embodiments, the stirring step comprises a duration of up to 48 hours. In some embodiments, the stirring step comprises a duration of up to 24 hours. In some embodiments, the stirring step comprises a duration of up to 18 hours. In some embodiments, the stirring step comprises a duration of up to 12 hours. In some embodiments, the stirring step comprises a duration of 12 to 24 hours (e.g., 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours). In some embodiments, the stirring step comprises a duration of 12 to 48 hours (e.g., 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours). In some embodiments, the stirring step comprises a duration of 24 to 48 hours.

[0065] In some embodiments, the agitation step has a duration equal to or less than the doubling time of the first population of cells and / or the second population of cells. In some embodiments, the agitation step has a duration equal to or less than 3 times (e.g., equal to or less than 2 times or equal to or less than 1 times) the doubling time of the first population of cells and / or the second population of cells. Such constraints may prevent the number of cells from being increased to promote aggregation without promoting proliferation. For example, if a population of cells has a doubling time of 24-36 hours, in some embodiments, the agitation step may not exceed 72 hours (i.e., not exceed 2 times the 24 hour doubling time).

[0066] In some embodiments, agitation can be accomplished by any suitable method known in the art, such as, for example, stirring, shaking, rotating, spinning at high speed, etc.

[0067] In some embodiments, the agitation comprises rotating the bioreactor at a speed between 10 RPM and 50 RPM (e.g., 15 RPM, 20 RPM, 25 RPM, 30 RPM, 35 RPM, 40 RPM, 45 RPM, or 50 RPM, e.g., 36 RPM).

[0068] In some embodiments, the bioreactor is incubated at a temperature between 35° C. and 39° C. (e.g., 35° C., 35.5° C., 36° C., 36.5° C., 37° C., 37.5° C., 38° C., 38.5° C., or 39° C.).

[0069] In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an average diameter within ±10% (e.g., ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of each other. In some embodiments, the aggregates are spheroids.

[0070] One of skill in the art will appreciate that a population of aggregates may have a range of Z-average mean particle diameters within the population. Thus, the population may be polydisperse. The population may have a polydispersity index of 0.7 or less (e.g., 0.05 to 0.7, e.g., 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.65, or 0.7). The polydispersity index may be measured using DLS (see, e.g., ISO22412:2017). Purification may be used to reduce the polydispersity of the population of aggregates produced by this method, e.g., to produce a more monodisperse population.

[0071] In some embodiments, the method produces aggregate densities of 500 aggregates / mL to 10,000 aggregates / mL (e.g., 500 aggregates / mL, 600 aggregates / mL, 700 aggregates / mL, 800 aggregates / mL, 900 aggregates / mL, 1,000 aggregates / mL, 2,000 aggregates / mL, 3,000 aggregates / mL, 4,000 aggregates / mL, 5,000 aggregates / mL, 6,000 aggregates / mL, 7,000 aggregates / mL, 8,000 aggregates / mL, 9,000 aggregates / mL, or 10,000 aggregates / mL).

[0072] In some embodiments, the method produces an average aggregate mean diameter of 50 μm to 200 μm (e.g., 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, or 200 μm). In some embodiments, the aggregates (e.g., at least 80%, e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates each have an average diameter of at least 50 μm (e.g., at least 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, or more).

[0073] In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an average diameter of less than 200 μm. In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an area / perimeter ratio of 45 or less, e.g., 40, 35, 30, 25, 20, 15, or 10 or less.

[0074] In some embodiments, each of the aggregates (e.g., at least 80%, e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates has a molecular weight of at least 1 × 10 3 cells (e.g., at least 1 x 10 3 cells, 2 x 10 3 cells, 3 x 10 3 cells, 4 x 10 3 cells, 5 x 10 3 cells, 6 x 10 3 cells, 7 x 10 3 cells, 8 x 10 3 cells, 9 x 10 3 cells, or 1 x 10 4 In some embodiments, the aggregates each contain at least 1 x 10 5 cells, 1 x 10 6 cells, 1 x 10 7 cells, or 1 x 10 8 Contains cells.

[0075] In some embodiments, the method produces an average total volume of aggregates of between 200 μL and 50 mL, e.g., between 200 μL and 1 mL (e.g., 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 550 μL, 600 μL, 650 μL, 700 μL, 750 μL, 800 μL, 850 μL, 900 μL, 950 μL, or 1 mL), between 1 mL and 10 mL (e.g., 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL), or between 10 mL and 100 mL (e.g., 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL).

[0076] In some embodiments, the method produces an average total mass of aggregates of between 100 mg and 100 g, e.g., between 100 mg and 1 g (e.g., 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1 g), between 1 g and 10 g (e.g., 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, or 10 g), or between 10 g and 100 g (e.g., 10 g, 20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, or 100 g).

[0077] The methods described herein may further include collecting the aggregates from the bioreactor. Collecting the aggregates may include, for example, pipetting, pouring, decanting, or draining the bioreactor. In some embodiments, the bioreactor includes a collection tube and the method includes collecting the aggregates in the collection tube.

[0078] In some embodiments, the method further comprises washing the collected aggregates, e.g., with a wash buffer, e.g., to remove any medium. Washing may include, e.g., pipetting, pouring, decanting, or draining the medium. Washing may include dialysis or exchange of the medium. The aggregates may be washed by centrifugation or acoustic separation. For example, the collected aggregates may be washed by countercurrent centrifugation or density gradient centrifugation. The aggregates may be washed by a CTS™ ROTEA™ countercurrent centrifugation system.

[0079] In some embodiments, the method further comprises washing the cells prior to culturing the cells in suspension, e.g., after thawing the cells. In some embodiments, the method further comprises purifying the collected aggregates. The collected aggregates may be purified, for example, by centrifugation or acoustic separation. For example, the collected aggregates may be purified by countercurrent centrifugation or density gradient centrifugation. In some embodiments, at least 80% (e.g., at least 80%, 85%, 90%, 95%, 97%, or 99%) of the aggregates have an average diameter of ±10% (e.g., ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of each other after purification.

[0080] In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an average diameter of less than 200 μm. In some embodiments, at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) of the aggregates have an area / perimeter ratio of 45 or less, e.g., 40, 35, 30, 25, 20, 15, or 10 or less.

[0081] The aggregates may be purified by a CTS ROTEA™ countercurrent centrifugation system. In some embodiments, the method further comprises concentrating the collected aggregates, for example to a concentration suitable for incorporation into a biocompatible scaffold.

[0082] In some embodiments, the method further comprises formulating the collected aggregates in a storage buffer, e.g., for storage or downstream use. Bioreactor The methods described herein include agitating a plurality of cell populations in a bioreactor, which may be any suitable bioreactor sufficient to agitate the cell populations and maintain the cells in suspension during agitation.

[0083] In some embodiments, the bioreactor has a volume of 0.1 L to 500 L, e.g., 0.1 L to 10 L (0.1 L, 0.2 L, 0.3 L, 0.4 L, 0.5 L, 0.6 L, 0.7 L, 0.8 L, 0.9 L, or 1 L), 1 L to 10 L (e.g., 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, or 10 L), 10 L to 100 L (e.g., 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, or 100 L), or 100 L to 500 L (e.g., 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 450 L, or 500 L). In some embodiments, the volume of the bioreactor is between 100 L and 300 L. In some embodiments, the volume of the bioreactor is 0.5 L.

[0084] The bioreactor used in the methods described herein may be a stirred tank bioreactor (e.g., a disposable stirred tank bioreactor) or a vertical wheel bioreactor. In some embodiments, the bioreactor is a BIOBLUE® c Single-Use Bioreactor (Eppendorf). In some embodiments, the bioreactor is a SCIVARIO® Twin Bioreactor (Eppendorf).

[0085] The bioreactor may further comprise a biotreatment controller, which may control one or more of pH, temperature, and dissolved oxygen concentration. In some embodiments, the first population of cells and the second population of cells do not adhere to the bioreactor, and therefore the bioreactor may include a coating to prevent cell adhesion.

[0086] In some embodiments, the bioreactor (e.g., a vertical wheel bioreactor) rotates at a speed of 10 RPM to 50 RPM (e.g., 15 RPM, 20 RPM, 25 RPM, 30 RPM, 35 RPM, 40 RPM, 45 RPM, or 50 RPM, e.g., 36 RPM).

[0087] In some embodiments, the bioreactor comprises a collection tube and the method includes collecting the aggregates in the collection tube. Flocculation medium The methods described herein include agitating a population of cells in a liquid medium to form aggregates. The liquid medium may be any suitable medium sufficient to form aggregates. The medium may, for example, include serum (e.g., human serum) or a serum replacement.

[0088] In some embodiments, the viscosity of the medium is 0.5 cP to 2 cP (e.g., 0.9 cP to 1.4 cP, e.g., 0.5 cP, 0.6 cP, 0.7 cP, 0.8 cP, 0.9 cP, 1 cP, 1.1 cP, 1.2 cP, 1.3 cP, 1.4 cP, 1.5 cP, 1.6 cP, 1.7 cP, 1.8 cP, 1.9 cP, or 2 cP). Viscosity may be adjusted to optimize cell-cell contact and enhance aggregation.

[0089] In some embodiments, the medium contains 1-20 μg / mL recombinant human insulin, 1-10 μg / mL human transferrin, and 1×10 -3 ~1×10 -2 Contains μg / mL of selenite.

[0090] In some embodiments, the medium contains laminin, collagen, elastin, or fibronectin. For example, the medium may contain 1 to 10 μg / mL (e.g., 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, or 10 μg / mL) of laminin.

[0091] In some embodiments, the medium contains 1 to 20 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM) of a Rho-associated protein kinase (ROCK) inhibitor. The ROCK inhibitor may be, for example, Y27632. Other ROCK inhibitors include, but are not limited to, AT-13148, BA-210, β-elemene, chroman 1, DJ4, fasudil, GSK-576371, GSK429286A, H-1152, hydroxyfasudil, LX-7101, netarsudil, RKI-1447, ripasudil, TCS-7001, thiazovivin, belosudil, Y-30141, Y33075, Y-39983.

[0092] In some embodiments, the medium comprises human serum. For example, the medium may comprise 0.1% to 20% (v / v) human serum, for example, 0.1% to 1% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), for example, 1% to 10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%), or 10% to 20% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some embodiments, the medium comprises 1% to 10% (v / v) human serum. In some embodiments, the medium comprises 5% (v / v) human serum. In other embodiments, the medium contains 10% (v / v) human serum.

[0093] In some embodiments, the medium comprises a platelet lysate (e.g., human platelet lysate, e.g., PLATELET GOLD™). For example, the medium may comprise 0.1%-10% (v / v) platelet lysate, e.g., 0.1%-1% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or 1%-10% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) platelet lysate. In some embodiments, the medium comprises 1%-5% (v / v) platelet lysate. In some embodiments, the medium comprises 5% (v / v) platelet lysate.

[0094] In some embodiments, the medium comprises glucose (e.g., 4,000 mg / L to 5,000 mg / L glucose, e.g., 4,100 mg / L, 4,200 mg / L, 4,300 mg / L, 4,400 mg / L, 4,500 mg / L, 4,600 mg / L, 4,700 mg / L, 4,800 mg / L, 4,900 mg / L, or 5,000 mg / L glucose). In some embodiments, the medium comprises 4,500 mg / L glucose.

[0095] In some embodiments, the medium comprises glucagon (e.g., 10 ng / mL to 100 ng / mL of glucagon, e.g., 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL of glucagon). In some embodiments, the medium comprises 40 ng / mL of glucagon.

[0096] In some embodiments, the medium comprises fibrinogen. In some embodiments, the medium comprises dexamethasone (e.g., 10 ng / mL to 100 ng / mL dexamethasone, e.g., 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL dexamethasone). In some embodiments, the medium comprises 60 ng / mL dexamethasone.

[0097] Cell populations The methods described herein include agitating a plurality of cell populations to produce aggregates. The cell populations may be optimized to maintain appropriate morphology, phenotype, and cell function that facilitates use in the methods of the present disclosure. The cell populations of the methods described herein may include a population of mammalian (e.g., human) cells. The populations may include primary cells, engineered cells, cell aggregates, induced pluripotent stem cell (iPSC)-derived cells, embryonic stem cell (ESC)-derived cells, transdifferentiated cells, or combinations thereof. The cells may be primary cells, such as, for example, primary cells grown in vitro. In some embodiments, the population of cells includes endocrine cells, exocrine cells, paracrine cells, heterocrine cells, autocrine cells, or juxtacrine cells.

[0098] In some embodiments, the population of cells comprises Leydig cells, adrenal cortical cells, pituitary cells, thyroid cells, granulosa cells, mammary epithelial cells, thymus cells, thymic epithelial cells, hypothalamic cells, skeletal muscle cells, smooth muscle cells, and / or neuronal cells.

[0099] In some embodiments, the pituitary cells comprise thyrotropin-producing pituitary cells, lactotropin-producing pituitary cells, adrenocorticotropin-producing pituitary cells, growth hormone-producing pituitary cells, and / or gonadotropin-producing pituitary cells.

[0100] In some embodiments, the neuronal cells comprise dopaminergic cells. In some embodiments, the population of cells comprises parenchymal cells (e.g., hepatocytes, pancreatic exocrine cells, muscle cells, pancreatic endocrine cells, neurons, intestinal cells, adipocytes, spleen cells, kidney cells, bile duct cells, Kupffer cells, stellate cells, cardiomyocytes, alveolar epithelial cells, bronchiolar cells, club cells, urothelial cells, mucus cells, parietal cells, chief cells, G cells, goblet cells, enteroendocrine cells, Paneth cells, M cells, tuft cells, glial cells, gallbladder cells, keratinocytes, melanocytes, Merkel cells, Langerhans cells, osteocytes, osteoclasts, esophageal cells, photoreceptor cells, and corneal epithelial cells). In some embodiments, the parenchymal cells are pancreatic cells (e.g., alpha cells, beta cells, gamma cells, delta cells, epsilon cells, or any combination thereof). In some embodiments, the parenchymal cells comprise beta cells.

[0101] In some embodiments, the method involves aggregating two or more populations of cells (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more populations of cells).

[0102] In some embodiments, the population of cells comprises a population of hepatic cells and a population of stromal cells. In some embodiments, the stromal cells comprise fibroblasts. In some embodiments, the fibroblasts are human dermal fibroblasts (e.g., normal human skin fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular fibroblasts, human atrial fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gallbladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts). In some embodiments, the fibroblasts are human skin fibroblasts. In some embodiments, the fibroblasts are normal human skin fibroblasts. In some embodiments, the fibroblasts are neonatal foreskin fibroblasts. In some embodiments, the fibroblasts are human lung fibroblasts. In some embodiments, the fibroblasts are human ventricular fibroblasts. In some embodiments, the fibroblasts are human atrial fibroblasts. In some embodiments, the fibroblasts are human uterine fibroblasts. In some embodiments, the fibroblasts are human bladder fibroblasts. In some embodiments, the fibroblasts are human gingival fibroblasts. In some embodiments, the fibroblasts are human pericardial fibroblasts. In some embodiments, the fibroblasts are human gallbladder fibroblasts. In some embodiments, the fibroblasts are human portal vein fibroblasts. In some embodiments, the fibroblasts are vas deferens fibroblasts.

[0103] In some embodiments, the cells are engineered cells, primary cells, or transdifferentiated cells. In some embodiments, the engineered cells are engineered to express or secrete a protein, such as an antibody, a cytokine, an enzyme, a clotting factor, or a hormone. The protein may be, for example, an endogenous human protein or an artificial protein.

[0104] In some embodiments, the first population of cells comprises stromal cells (e.g., fibroblasts). The fibroblasts may be, for example, primary fibroblasts, induced pluripotent stem cell (iPSC)-derived fibroblasts, or embryonic stem cell (ESC)-derived fibroblasts. In some embodiments, the fibroblasts are genetically engineered fibroblasts.

[0105] In some embodiments, the second population of cells comprises parenchymal cells. The parenchymal cells may be, for example, hepatocytes (e.g., human hepatocytes, e.g., primary human hepatocytes) or hepatocyte progenitor cells. The hepatocytes may be, for example, primary hepatocytes (e.g., primary human hepatocytes), iPSC-derived hepatocytes, or ESC-derived hepatocytes. The hepatocytes may be genetically engineered hepatocytes. The parenchymal cells may comprise pancreatic cells (e.g., alpha cells, beta cells, gamma cells, delta cells, or epsilon cells, or combinations thereof) or pancreatic progenitor cells. The pancreatic cells may be, for example, primary human pancreatic cells, iPSC-derived pancreatic cells, or ESC-derived pancreatic cells. In some embodiments, the pancreatic cells are genetically engineered pancreatic cells.

[0106] In some embodiments, the first population of cells comprises fibroblasts and the second population of cells comprises hepatocytes. In some embodiments, the first population of cells comprises fibroblasts and the second population comprises hepatocyte progenitor cells.

[0107] In some embodiments, the first population of cells comprises fibroblasts and the second population comprises pancreatic beta cells. In some embodiments, the ratio of the first population of cells (e.g., stromal cells, e.g., fibroblasts, e.g., normal human dermal fibroblasts) to the second population of cells (e.g., parenchymal cells, e.g., hepatocytes, e.g., primary human hepatocytes) is 10:1 to 1:10 (e.g., 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 1:10 to 1:1 to 1:2, 1:1 to 1:3, 1:1 to 1:4, 1:1 to 1:5, 1:1 to 1:6, 1:1 to 1:7, 1:1 to 1:8, 1:1 to 1:9, or 1:1 to 1:10). For example, the ratio of the first population of cells to the second population of cells can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the ratio is 2:1.

[0108] In some embodiments, the first population of cells (e.g., stromal cells, e.g., fibroblasts, e.g., normal human dermal fibroblasts) and the second population of cells (e.g., parenchymal cells, e.g., hepatocytes, e.g., primary human hepatocytes) do not expand (e.g., in cell number) by more than 30% during the agitating step. For example, in some embodiments, the first population of cells and the second population of cells do not expand by more than 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0109] In some embodiments, the method promotes aggregation without causing a substantial increase in cell number. For example, in some embodiments, the ratio of the first population of cells to the second population of cells does not increase by more than 10-fold (e.g., does not increase by more than 9.5-fold, 9-fold, 8-fold, 7.5-fold, 7-fold, 6.5-fold, 6-fold, 5.5-fold, 5-fold, 4.5-fold, 4-fold, 3.5-fold, 3-fold, 2.5-fold, 2-fold, 1.5-fold, or 1-fold). For example, in some embodiments, the initial ratio of the first population of cells to the second population of cells (e.g., NHDF:PHH) is 2:1, and this ratio does not increase by more than 4:1 or 5:1 (i.e., does not increase by more than 2.5-fold).

[0110] In some embodiments, the ratio of the first population of cells to the second population of cells is increased by 1-10-fold (e.g., 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold, e.g., 1-fold to 3-fold, e.g., 1-fold to 2.5-fold, e.g., 1-fold, 1.5-fold, 2-fold, or 2.5-fold).

[0111] Fibroblasts may have a doubling time of 24 to 36 hours. Thus, in some embodiments, the aggregation time of 24 to 36 hours (e.g., 24 hours) is equal to or less than the doubling time of fibroblasts, thus preventing any increase in cell number during aggregation. Similarly, PHHs have very long doubling times, e.g., approaching infinity, thus preventing any increase in cell number of PHHs during aggregation.

[0112] In some embodiments, the ratio of the first population of cells (e.g., stromal cells, e.g., fibroblasts, e.g., normal human dermal fibroblasts) to the second population of cells (e.g., parenchymal cells, e.g., hepatocytes, e.g., primary human hepatocytes) is between 0.1:1 and 5:1. In some embodiments, the ratio is between 0.2:1 and 3.8:1. In some embodiments, the ratio is 3.8:1. In some embodiments, the ratio is 3:1. In some embodiments, the ratio is 2.5:1. In some embodiments, the ratio is 2:1. In some embodiments, the ratio is 1:1. In some embodiments, the ratio is 0.2:1. In some embodiments, the first population of cells is between 1×10 4 cells / mL~1×10 8 The first population of cells is present at a density of 1 x 10 cells / mL. For example, the first population of cells is present at a density of 1 x 10 4 cells / mL~1×10 5 cells / mL (e.g., 1 x 10 4 cells / mL, 2×10 4 cells / mL, 3×10 4 cells / mL, 4×10 4 cells / mL, 5×10 4 cells / mL, 6×10 4 cells / mL, 7×10 4 cells / mL, 8×10 4 cells / mL, 9×10 4 cells / mL, or 1 x 10 5 cells / mL), 1×10 5 cells / mL~1×10 6 cells / mL (e.g., 1 x 10 5 cells / mL, 2×10 5 cells / mL, 3×10 5cells / mL, 4×10 5 cells / mL, 5×10 5 cells / mL, 6×10 5 cells / mL, 7×10 5 cells / mL, 8×10 5 cells / mL, 9×10 5 cells / mL, or 1 x 10 6 cells / mL), 1×10 6 cells / mL~1×10 7 cells / mL (e.g., 1 x 10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, or 1 x 10 7 cells / mL), or 1 × 10 7 cells / mL~1×10 8 cells / mL (e.g., 1 x 10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, 4×10 7 cells / mL, 5×10 7 cells / mL, 6×10 7 cells / mL, 7×10 7 cells / mL, 8×10 7 cells / mL, 9×10 7 cells / mL, or 1 x 10 8 The cells may be present at a density of 1000 x 1000 cells / mL.

[0113] In some embodiments, the first population of cells comprises fibroblasts (e.g., primary fibroblasts, e.g., primary human fibroblasts), and the fibroblasts have a density of 1×10 5 cells / mL~1×10 7 cells / mL (e.g., 1 x 10 5 cells / mL, 2×10 5 cells / mL, 3×10 5 cells / mL, 4×10 5cells / mL, 5×10 5 cells / mL, 6×10 5 cells / mL, 7×10 5 cells / mL, 8×10 5 cells / mL, 9×10 5 cells / mL, 1×10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, or 1 x 10 7 For example, the density of fibroblasts is, for example, 6 × 10 5 It may be in cells / mL.

[0114] In some embodiments, the second population of cells is 1×10 4 cells / mL~1×10 8 For example, the second population of cells is present at a density of 1 x 10 4 cells / mL~1×10 5 cells / mL (e.g., 1 x 10 4 cells / mL, 2×10 4 cells / mL, 3×10 4 cells / mL, 4×10 4 cells / mL, 5×10 4 cells / mL, 6×10 4 cells / mL, 7×10 4 cells / mL, 8×10 4 cells / mL, 9×10 4 cells / mL, or 1 x 10 5 cells / mL), 1×10 5 cells / mL~1×10 6 cells / mL (e.g., 1 x 10 5 cells / mL, 2×10 5 cells / mL, 3×10 5 cells / mL, 4×10 5 cells / mL, 5×10 5 cells / mL, 6×10 5cells / mL, 7×10 5 cells / mL, 8×10 5 cells / mL, 9×10 5 cells / mL, or 1 x 10 6 cells / mL), 1×10 6 cells / mL~1×10 7 cells / mL (e.g., 1 x 10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, or 1 x 10 7 cells / mL), or 1 × 10 7 cells / mL~1×10 8 cells / mL (e.g., 1 x 10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, 4×10 7 cells / mL, 5×10 7 cells / mL, 6×10 7 cells / mL, 7×10 7 cells / mL, 8×10 7 cells / mL, 9×10 7 cells / mL, or 1 x 10 8 They may be present at a density of cells / mL.

[0115] In some embodiments, the second population of cells comprises hepatocytes (e.g., primary hepatocytes, e.g., primary human hepatocytes), and the density of the hepatocytes is 1×10 5 cells / mL~1×10 7 cells / mL (e.g., 1 x 10 5 cells / mL, 2×10 5 cells / mL, 3×10 5 cells / mL, 4×10 5 cells / mL, 5×10 5 cells / mL, 6×10 5 cells / mL, 7×10 5 cells / mL, 8×105 cells / mL, 9×10 5 cells / mL, 1×10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, or 1 x 10 7 For example, the density of hepatocytes is, for example, 3 × 10 5 It may be in cells / mL.

[0116] In some embodiments, the total number of cells (e.g., viable cells) is greater than or equal to 1×10 4 cells / mL~1×10 8 For example, the second population of cells is present at a density of 1 x 10 4 cells / mL~1×10 5 cells / mL (e.g., 1 x 10 4 cells / mL, 2×10 4 cells / mL, 3×10 4 cells / mL, 4×10 4 cells / mL, 5×10 4 cells / mL, 6×10 4 cells / mL, 7×10 4 cells / mL, 8×10 4 cells / mL, 9×10 4 cells / mL, or 1 x 10 5 cells / mL), 1×10 5 cells / mL~1×10 6 cells / mL (e.g., 1 x 10 5 cells / mL, 2×10 5 cells / mL, 3×10 5 cells / mL, 4×10 5 cells / mL, 5×10 5 cells / mL, 6×10 5 cells / mL, 7×10 5 cells / mL, 8×10 5 cells / mL, 9×10 5cells / mL, or 1 x 10 6 cells / mL), 1×10 6 cells / mL~1×10 7 cells / mL (e.g., 1 x 10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, or 1 x 10 7 cells / mL), or 1 × 10 7 cells / mL~1×10 8 cells / mL (e.g., 1 x 10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, 4×10 7 cells / mL, 5×10 7 cells / mL, 6×10 7 cells / mL, 7×10 7 cells / mL, 8×10 7 cells / mL, 9×10 7 cells / mL, or 1 x 10 8 In some embodiments, the total number of cells (e.g., viable cells) may be present at a density of 1×10 5 cells / mL~1×10 7 cells / mL (e.g., 3 x 10 5 cells / mL~1.5×10 6 In some embodiments, the total number of cells (e.g., viable cells) is present at a density of 1.5×10 6 Present at a density of cells / mL.

[0117] How to pack The aggregates produced by the methods described herein may further be encapsulated within a scaffold (e.g., a biocompatible scaffold). Such encapsulated cell scaffolds may be used to form grafts (e.g., tissue grafts) for various downstream applications, such as implantation into a subject (e.g., a human subject). The method includes providing a population of aggregates of a plurality of cell populations (e.g., a first cell population and a second cell population) and a biocompatible scaffold. The method may further include introducing a polymerization agent or crosslinking reagent to polymerize or crosslink the biocompatible scaffold, thereby encapsulating the population of aggregates, for example, within the biocompatible scaffold. In other examples, the method may include incubating the aggregates for a time and under conditions sufficient to result in polymerization of the biocompatible scaffold without adding a polymerization agent or crosslinking reagent. The aggregates may include, for example, two or more populations of cells (e.g., a population of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cells). The aggregates may include, for example, a first population of cells and a second population of cells.

[0118] Biocompatible Scaffolds The aggregates described herein may be encapsulated within a biocompatible scaffold containing cells, such as hepatocytes and stromal cells. In some embodiments, the population of cells (e.g., hepatocyte and stromal cell populations) aggregate into spheroids. In some embodiments, the biocompatible scaffold has an x-axis, a y-axis, and a z-axis. For example, in some embodiments, the population of cells, such as hepatocytes and optional stromal cell populations, aggregate into spheroids, and the spheroids are distributed non-homogenously in layers along the z-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogeneously along the x-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogeneously along the y-axis of the biocompatible scaffold.

[0119] A biocompatible scaffold may be liquid, gel, semi-solid, or solid at room temperature (e.g., 25° C.). A biocompatible scaffold may be biodegradable or non-biodegradable. In some embodiments, the scaffold is bioabsorbable or bioreplaceable. Exemplary biocompatible scaffolds include polymers and hydrogels, examples of which include collagen, fibrinogen, fibrin, chitosan, MATRIGEL™, dextran, including chemically crosslinkable or photocrosslinkable dextran, engineered tissue matrices such as submucosa, PEG hydrogels (e.g., heparin-conjugated PEG hydrogels), poly(lactic-co-glycolic acid) (PLGA), hydroxyethyl methacrylate (HEMA), gelatin, alginate, agarose, polysaccharides, hyaluronic acid (HA), peptide-based self-assembling gels, and thermoresponsive poly(NIPAAm). Many biopolymers are known to those of skill in the art (Bryant and Anseth, J. Biomed. Mater. Res. (2002) 59(1):63-72; Mann et al., Biomaterials (2001) 22(22):3045-3051; Mann et al., Biomaterials (2001) 22(5):439-444, and Peppas et al., Eur. J. Pharm. Biopharm. (2000) 50(1), 27-46; all incorporated by reference.) In other embodiments, the biocompatible scaffold may contain biopolymers that include any of a variety of growth factors, adhesion molecules, degradation sites, or bioactive agents to enhance cell viability, or for any of a variety of other reasons. Such molecules are well known to those of skill in the art.

[0120] In some embodiments, the PEG hydrogel may be chemically crosslinkable and / or modified with bifunctional groups. In certain embodiments, the biocompatible scaffold comprises allogeneic components, autologous components, or both allogeneic and autologous components. In certain embodiments, the biocompatible scaffold comprises a synthetic or semi-synthetic material. In certain embodiments, the biocompatible scaffold comprises a framework or support, such as a fibrin-derived scaffold.

[0121] In some embodiments, the biocompatible scaffold comprises fibrin. In some embodiments, the polymerization agent comprises thrombin. After the polymerization agent (such as thrombin) is introduced, fibrinogen within the scaffold is polymerized into fibrin.

[0122] In some embodiments, the biocompatible scaffold further comprises a reinforcing agent, for example, the reinforcing agent may include collagen, poly(ethylene glycol), polyvinylidene acetate (PVDA), polyvinylidene fluoride (PVDF), poly(lactic-co-glycolic acid) (PLGA), or poly(L-lactic acid) (PLLA).

[0123] Biocompatible hydrogel scaffolds suitable for use include any polymer capable of gelling in situ (e.g., polymers that do not require non-cytocompatible chemicals or conditions (e.g., temperature or pH). This includes both stable and biodegradable biopolymers.

[0124] The polymers used herein are preferably crosslinked, for example ionically crosslinked. In certain embodiments, the methods and constructs described herein use polymers that can be photochemically (i.e., photocrosslinked) by exposure to light of an appropriate wavelength to promote polymerization (i.e., photopolymerizable), or that can be weakened or soluble by exposure to light or another stimulus. Some of the polymers listed above are not inherently light sensitive (e.g., collagen, HA), but can be made light sensitive by adding acrylate or other photosensitive groups.

[0125] In certain embodiments, the method utilizes a photoinitiator. A photoinitiator is a molecule that can promote polymerization of a hydrogel when exposed to the appropriate wavelength of light defined by the reactive groups on the molecule. In the context of the present disclosure, the photoinitiator is cytocompatible. There are many known photoinitiators that can be used with various wavelengths of light. For example, 2,2-dimethoxy-2-phenyl-acetophenone, HPK 1-hydroxycyclohexyl-phenyl ketone, and Irgacure 2959 (hydroxyl-1-[4-(hydroxyethoxy)phenyl]-2 methyl-1 propanone) are all activated by UV light (365 nm). Other crosslinkers that are activated by cytocompatible wavelengths of light (e.g., blue light) can also be used in the methods described herein.

[0126] In other embodiments, the method involves the use of a polymer having a moiety that is not photochemically polymerizable. In certain embodiments, the moiety that is not photochemically polymerizable is a Michael acceptor. Non-limiting examples of such Michael acceptor moieties include α,β-unsaturated ketones, esters, amides, sulfones, sulfoxides, phosphonates. Additional non-limiting examples of Michael acceptors include quinine and vinylpyridine. In some embodiments, polymerization of the Michael acceptor is facilitated by a nucleophile. Suitable nucleophiles include, but are not limited to, thiols, amines, alcohols, and molecules having thiol, amine, and alcohol moieties. In certain embodiments, the present disclosure features the use of thermally crosslinked polymers.

[0127] In some embodiments, the z-axis of the biocompatible scaffold is between 500 μm and 5 mm (e.g., between 600 μm and 4 mm, between 700 μm and 3 mm, between 800 μm and 2 mm, or between 900 μm and 1 mm). For example, in some embodiments, the z-axis of the biocompatible scaffold is between 600 μm and 4 mm. In some embodiments, the z-axis of the biocompatible scaffold is between 700 μm and 3 mm. In some embodiments, the z-axis of the biocompatible scaffold is between 800 μm and 2 mm. In some embodiments, the z-axis of the biocompatible scaffold is between 900 μm and 1 mm.

[0128] In some embodiments, the z-axis of the biocompatible scaffold is 500 μm. In some embodiments, the z-axis of the biocompatible scaffold is 600 μm. In some embodiments, the z-axis of the biocompatible scaffold is 700 μm. In some embodiments, the z-axis of the biocompatible scaffold is 800 μm. In some embodiments, the z-axis of the biocompatible scaffold is 900 μm. In some embodiments, the z-axis of the biocompatible scaffold is 1 mm. In some embodiments, the z-axis of the biocompatible scaffold is 2 mm. In some embodiments, the z-axis of the biocompatible scaffold is 3 mm. In some embodiments, the z-axis of the biocompatible scaffold is 4 mm. In some embodiments, the z-axis of the biocompatible scaffold is 5 mm.

[0129] In some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer of cell aggregates (e.g., hepatocyte and stromal cell aggregates) within the biocompatible scaffold is 20:1 to 1:1 (e.g., 19:1 to 1:1, 18:1 to 1:1, 17:1 to 1:1, 16:1 to 1:1, 15:1 to 1:1, 14:1 to 1:1, 13:1 to 1:1, 12:1 to 1:1, 11:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, or 2:1 to 1:1). For example, in some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer is 19:1 to 1:1. In some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer is between 18:1 and 1:1. In some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer is between 17:1 and 1:1. In some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer is between 16:1 and 1:1. In some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer is between 15:1 and 1:1. In some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer is between 14:1 and 1:1. In some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer is between 13:1 and 1:1. In some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer is between 12:1 and 1:1. In some embodiments, the ratio of the height of the biocompatible scaffold to the height of the layer is between 11:1 and 1:1. In some embodiments, the ratio of the biocompatible scaffold height to the layer height is between 10:1 and 1:1. In some embodiments, the ratio of the biocompatible scaffold height to the layer height is between 9:1 and 1:1. In some embodiments, the ratio of the biocompatible scaffold height to the layer height is between 8:1 and 1:1. In some embodiments, the ratio of the biocompatible scaffold height to the layer height is between 7:1 and 1:1.In some embodiments, the ratio of the biocompatible scaffold height to the layer height is between 6:1 and 1:1. In some embodiments, the ratio of the biocompatible scaffold height to the layer height is between 5:1 and 1:1. In some embodiments, the ratio of the biocompatible scaffold height to the layer height is between 4:1 and 1:1. In some embodiments, the ratio of the biocompatible scaffold height to the layer height is between 3:1 and 1:1.

[0130] In some embodiments, the biocompatible scaffold comprises a synthetic heparin mimetic. In particular, the synthetic polymers of the present disclosure may, in some embodiments, comprise an amount of negative charges similar to the amount of negative charges present in heparin. Thus, the synthetic polymers of the present disclosure can mimic the functional properties of heparin. For example, the synthetic polymers of the present disclosure have the potential to bind various bioactive agents that naturally bind to heparin, such as growth factors. Thus, the synthetic polymers of the present disclosure, and hydrogels comprising the synthetic polymers described herein, can bind various bioactive agents, such as growth factors, thereby preventing the bioactive agent from diffusing and maintaining a high concentration of the bioactive agent locally, which can act on cells and promote various cellular functions. EXAMPLES

[0131] Example 1. Production of aggregates in a bioreactor VWB_005 Survey These experiments were performed to evaluate the aggregation of normal human dermal fibroblasts (NHDF) and primary human hepatocytes (PHH) in a vertical wheel bioreactor platform when seeded at the same concentrations as in a microwell platform that can be used to form aggregates. The effects of ROCK inhibitor, fibrinogen, and laminin on the aggregation of NHDF and PHH in the VWB platform were evaluated.

[0132] Testing Group: 1. Only NHDF+PHH in ITS @ 36RPM (volume 75mL) 2. NHDF+PHH+10 μM ROCK inhibitor in ITS @ 36 RPM (volume 75 mL) 3. NHDF+PHH+1mg / mL FIBRYGA (registered trademark)@36RPM (volume 75mL) in ITS 4. NHDF+PHH+5μg / mL laminin in ITS @36RPM (volume 75mL) NHDF: seeded at 6e5 million cells / mL Seeding at PHH:3.4e5 million cells / mL A summary of the experimental conditions utilized is provided below.

[0133] 1. NHDF and PHH were mixed together by adding an equal volume of PHH solution to the NHDF solution in a 500mL conical. For example, if there was 100mL of NHDF, add 100mL of PHH. This will give a 1.5x solution of NHDF+PHH. Target total mixed volume: 200mL minimum. 2. 50 mL of the mixture of NHDF and PHH 1.5x was added to each PBS-MINI® chamber. ROCK inhibitor, fibrinogen concentration (human), FIBRYGA®, or laminin was added to the corresponding chamber. The final volume was brought up to 75 mL using ITS medium.

[0134] 3. The chambers were transferred to bases placed inside the incubator. Each base was set at 36 RPM. The mean aggregate diameter and aggregate number were very similar in the ITS, laminin, and ROCK inhibitor conditions, but differences emerged in the debris and superaggregate numbers (Figure 1A, Figure 1B, Figure 2A-2D, Figure 3, and Figure 4). The ROCK inhibitor condition produced the least debris compared to the ITS and laminin conditions. In terms of superaggregate number, all three conditions produced low numbers (≦3), but the laminin condition produced the most superaggregates.

[0135] Superaggregates were identified as optically opaque structures with sizes greater than 200 μm and area / perimeter ratios greater than 45. As shown in Figures 5A-5D and 6, aggregates formed after 24 and 48 hours (Figure 6). After 48 hours, the number of aggregates decreased. The microwell baseline condition is a comparative condition in which cells aggregated within the microwells. In the microwell condition, more debris and superaggregates were produced (Figures 5A-5D).

[0136] The VWB_005 study showed that seeding of NHDFs and PHHs onto the VWB platform resulted in the formation of aggregates (also called seeds) in the presence of ITS, laminin (5 μg / mL), and ROCK inhibitor (10 μM).

[0137] VWB_006 Survey The efficacy of the seeds was then assessed by an ammonia clearance assay (IDEXX) and an albumin secretion assay.

[0138] [Table 1]

[0139] Seeding the bioreactor with NHDF and PHH 1. NHDF and PHH were mixed together by adding an equal volume of PHH solution to the NHDF solution in a 500 mL conical. Target total mixed volume: 100 mL minimum. 67.4 mL of each cell mixture was mixed.

[0140] 2. 50 mL of the mixture of NHDF and PHH 1.5x was added to both PBS-MINI® chambers. ROCK inhibitor was added to the designated chamber. The final volume was brought up to 75 mL using ITS medium.

[0141] 3. The chambers were transferred to bases placed inside the incubator. Each base was set at 36 RPM. Seeds were formed with ITS alone or in the presence of ROCK inhibitors, as shown in Figures 7A-7D, 10, 11, and 12. In ammonia clearance assays, these aggregates were able to reduce ammonia (Figures 8A and 8B) and showed comparable results to microwell-based aggregates (Figures 9A and 9B).

[0142] In conclusion, aggregates or seeds were generated in the VWB platform, demonstrating that suspension culture for aggregation is indeed possible. Although the VWB seeds were smaller than those generated in the microwell platform, they also produced significantly less debris and superaggregates compared to the microwell platform.

[0143] The 24-hour aggregation time was more successful than the 48-hour aggregation time. In this experiment, 48 hours appeared to be too long, resulting in significant superaggregate formation. Of the conditions tested, ITS, laminin, and ROCK inhibitor conditions all produced positive and similar results, with the ROCK inhibitor condition producing the least amount of debris after 24 hours.

[0144] VWB_007 Investigation VWB_007 was used to evaluate whether aggregation could occur similarly to VWB_005 and VWB_006 using alternative media formulations suitable for Good Manufacturing Practice (GMP) grade. The first objective was to remove the animal-derived component currently present in the aggregation media, fetal bovine serum (FBS). In this study, three concentrations of human serum and two concentrations of human platelet lysate were tested and compared to basal media conditions. All media contain high levels of glucose. As cells may be more stressed in conditions with low serum concentrations, ROCK inhibitor (ROCKi) was used in all conditions in this study.

[0145] [Table 2]

[0146] Seeding the bioreactor with NHDF and PHH For each condition: A. NHDF and PHH were mixed together by adding an equal volume (20 mL) of NHDF solution to PHH solution (20 mL) in a 50 mL conical for PHH. Mix the cells by pipetting up and down thoroughly.

[0147] B. The NHDF and PHH cell mixture was transferred to a compatible PBS-MINI® 0.1 L consumable (40 mL total). C. The final volume was brought up to 70 mL.

[0148] The chambers were transferred to bases which were placed in an incubator. Each base was set at 36 RPM. Similar to results seen in previous studies, the live cell concentration decreased over time while the dead cell concentration remained relatively constant. This indicates that single cells are incorporating into aggregates over time and are not dying (Figures 13A and 13B). Figure 13A shows live cells and Figure 13B shows dead cells. As shown in Figures 14A-14D, 15A-15D, 16, and 17, aggregates were formed under various conditions using human serum (HS) and PLATELET GOLD™ (PG, human platelet lysate), and the aggregate size, concentration, average volume, and total volume data were comparable to conditions without human serum or platelet lysate (Figures 14A-14D, 15A-15D, 16, and 17).

[0149] Aggregate Washing Individual vials of fibroblasts and hepatocytes were thawed separately and resuspended in cell culture medium prior to a centrifugation-based washing step. After aggregation, the aggregates were concentrated using a centrifuge and formulated in a fibrinogen solution for encapsulation. In this experiment, the CTS ROTEA™ counter-flow centrifugation system was evaluated as a viable option for post-thaw single cell washing, as well as post-aggregation seed washing and formulation.

[0150] Thawing and washing of NHDF A test of thawing and washing of NHDF cells was performed. Approximately 40M viable cells used corresponds to a real minimum wash run in our aggregation framework, i.e., a single PBS-MINI® 0.1 disposable vertical wheel bioreactor with 6e5 / mL NHDF cells. 35mL of cells in NHDF medium were bagged and the bag was coupled to the ROTEA™ port, and 47mL was harvested in a different bag. The harvest set point was 50mL, and the missing volume can be attributed to the dead volume of the tubing that was uncoupled but not collected (a 3mm inner diameter tubing occupies 3.2mL in 45cm). Viability after harvest was 96%, and 87% of viable cells were recovered after washing. These results show that a countercurrent centrifugation system such as ROTEA™ can be used to wash NHDF with minimal loss of viability.

[0151] The second role of the ROTEA™ experiment is the washing of the PHH. The viability of the PHH suspension was measured before bagging, at the harvest bag, and in the waste bag. Three tests were performed using the same ratio of flow rate to centrifuge speed, but varying the flow rate to vary the shear stress acting on the PHH.

[0152] Flow rate is a factor that can be altered to improve the overall viability of the crop. Figure 18 shows the number of live and dead cells (normal human dermal fibroblasts) after washing when thawed or washed with CTS™ ROTEA™ harvest using a counterflow centrifugation system. The number of live and dead PHH was dependent on different flow rates of the washing medium, as shown in Figures 19A-19C, 20A, and 20B.

[0153] Seed washing and formulation The aggregate washing and formulation trial consisted of two separate days of experiments. On the first day, aggregates were prepared using a microwell approach and pooled for processing. The processing was divided into two steps. First, the seeds were placed in the fluidized bed and returned to the first bag for sample harvest. This is close to a simple wash recovery. Second, the buffer was exchanged to a fibrinogen concentration of 14 mg / mL (human) (FIBRYGA®) with the aim of harvesting the concentrate. Two distinct populations of seeds and a debris count of over 200,000 were noticed.

[0154] The second day of seed washing used aggregates from VWB_007. The aggregates were pooled into two batches. Since the seeds generated in the suspension format are smaller than those generated in the microwell format, the instrument settings of the CTS™ ROTEA™ counter-current centrifugation system were adjusted accordingly.

[0155] Batch 1: ITS, 1% human serum, 5% Platelet Gold A pool of ITS, 1% human serum, and 5% Platelet Gold seeds was measured using a customized application. In this test, the system output 7 mL of liquid (10 mL target, 3 mL air) into a syringe coupled to the harvest line. From a starting reactor harvest volume of 250 mL, a 35-fold concentration was obtained.

[0156] After concentration and harvesting, the seeds were diluted 40-fold and re-measured. Compared to the initial conditions, the size range of the seeds became narrower and, surprisingly, larger aggregates were lost. ROTEA™ is able to remove the debris and generate a more monodisperse aggregate population (Figures 21 and 23).

[0157] Batch 2: 1% Platelet Gold, 5% human serum, 10% human serum In the second batch, the flow rate was increased in an attempt to achieve higher purification and a more uniform bed. The harvest volume set point was reduced to 5 mL. Pools of 1% Platelet Gold, 5% human serum, and 10% human serum reactors from VWB007 were measured.

[0158] These results, as shown in Figures 22 and 24, demonstrate that ROTEA™ can be used to wash the aggregates (seeds). VWB_010 Survey The VWB_005 study above demonstrated that the VWB system can produce seeds. In the VWB_006 study above, seeds were encapsulated and their functionality was demonstrated by ammonia clearance analysis. After it was demonstrated that the VWB system can produce functional seeds, this study assessed whether the method could be translated to Good Manufacturing Practice (GMP) standards, specifically using GMP media formulations. In previous studies, the aggregation medium was ITS, which contains animal-derived (non-human) supplements including fetal bovine serum (FBS), and various other components from the ITS premix. In this study, a GMP-aligned medium was formulated and produced that does not contain any non-GMP-compliant components. Replacing the ITS and FBS components with xeno-free reagents eliminates any potential risk of retaining animal-derived components while ensuring good health of the cells involved in the exemplary processes described herein. Thus, human serum (HS) and platelet lysate (e.g., Platelet Gold (PG)), which are produced and packaged according to GMP standards, were investigated.

[0159] In the VWB_007 study mentioned above, clumping was tested comparing media made with various percentages of human serum (HS) and Platelet Gold (PG) with a media formulation containing 10% fetal bovine serum. Seeds formed in all conditions tested, supporting a shift to non-bovine source serum in the clumping media. VWB_010 tested a subset of the media formulations queried in VWB_007, specifically aimed at understanding the in vitro function of the seeds to remove ammonia.

[0160] NHDF and PHH were thawed from cryopreservation, counted, and seeded at the indicated concentrations in a ratio of 2 NHDF to 1 PHH in PBS MINI 0.1 L bioreactors. Four media conditions were tested: ITS (10% FBS), 10% human serum (HS), 5% HS, and 5% Platelet Gold (PG). Suspension cultures were allowed to run for 24 h before harvesting.

[0161] Measurements included microscopy, seed number analysis, lactate dehydrogenase (LDH), alanine aminotransferase (ALT) / aspartate aminotransferase (AST), and in vitro graft efficacy (ammonia clearance).

[0162] The primary goals of this study were to evaluate the aggregation of NHDF and PHH on the VWB platform when seeded in various media formulations, to evaluate the effect of changing serum type to a xeno-free reagent on the VWB aggregation process, and to evaluate the efficacy and functionality of seeds aggregated on the VWB platform using xeno-free aggregation media.

[0163] Microscopy and seed counts Images were taken 24 hours after suspension-facilitated aggregation. Samples were diluted 4 times their initial volume for analysis by computational analysis. Figures 25A-25D show the results after computational analysis, including seed number, size, concentration, average seed volume (single seed), and total seed volume (volume fraction of seeds based on theoretical size).

[0164] Across the four media conditions, seed counts were relatively similar at around 2000-2200 seeds, with the 5% PG seeds showing a higher number (Figure 25A). The seed count was converted to seed concentration and counted per mL of reactor suspension. Again, the 5% PG condition was higher, meaning there were more seeds per mL compared to the other three conditions (Figure 25B). While the ITS and human serum conditions produced similar sized seeds, the 5% PG condition produced smaller seeds on average across all groups tested (Figure 25C). These data complement previous findings in Figure 25B. The 5% PG condition had smaller seeds, and therefore more seeds per mL of suspension. Seed size and debris are related. Conditions with larger seeds usually have less debris. This was seen for the ITS and 10% HS conditions (Figure 25D). Surprisingly, the seeds formed using 5% HS were slightly smaller, but also resulted in less debris. 5% PG produced the most debris under the conditions tested. Superaggregates were highest in the 10% human serum condition, as shown in Figure 26.

[0165] The mean seed diameter was very similar between the ITS and 10% HS groups, with 10% HS producing larger seeds on average. The 5% HS and 5% PG groups produced smaller seeds, with 5% PG producing the smallest seeds of all groups. As expected, the mean seed volume also followed a similar pattern and order. Total seed yield (seed volume / suspension volume) was similar for the ITS, 10% HS, and 5% PG groups (Figures 27A and 27B).

[0166] These data suggest that the higher concentration of human serum produced seeds similar to the control condition, the ITS group, in terms of number, size, debris, and volume, whereas the less concentrated HS condition produced smaller seeds.

[0167] Figures 28A and 28B show the cumulative amount of LDH calculated for each vertical wheel reactor (1x vertical wheel reactor) and encapsulated seeds (n=4 grafts per medium condition) during aggregation (Figure 28A).

[0168] After 24 h of aggregation, the 10% HS group produced the least amount of LDH, followed by ITS and 5% PG, whereas the 5% HS group appeared to produce the most amount of LDH at the 0 and 24 h time points.

[0169] During encapsulation, the 5% PG group produced the highest amount of LDH, the 5% HS group produced the lowest amount, and the ITS and 10% HS groups appeared to produce similar results.

[0170] Correction of these values ​​was achieved by subtracting the values ​​from the cell-free samples. Figures 30A and 30B show the ALT (Figure 29A) and AST (Figure 29B) values ​​produced during the 24-hour aggregation period for the four different media groups. Of the four media groups tested, the 10% HS group had the lowest ALT and AST levels during the 24-hour aggregation period. The ALT / AST levels in the 10% HS group were approximately half of the levels seen in the ITS, 5% HS, and 5% PG conditions. This suggests that primary human hepatocytes in the vertical wheel bioreactor are subjected to less stress in the 10% HS formulation during the aggregation process compared to the control group (ITS) and the other test groups.

[0171] All media formulations tested produced seeds capable of removing ammonia in an in vitro ammonia clearance assay. The performance of each group was very similar, with the 10% human serum group achieving the highest ammonia clearance of all groups tested. These data indicate that switching the serum source in the agglutination medium to a xeno-free one does not reduce the ammonia clearance ability of the seeds (Figures 30A and 30B).

[0172] In terms of overall ammonia clearance at 72 hours, the ITS, 5% HS, and 5% PG conditions performed similarly, with the 10% HS condition showing the highest level of overall relative ammonia clearance (Figure 31).

[0173] Looking at the k values, the ITS condition had the lowest k value, while the 10% HS condition had the highest k value. The 10% HS condition showed the highest k value, suggesting that this condition removed ammonia at a faster rate compared to the other groups (Figure 32).

[0174] In summary, the 10% human serum condition produced the lowest ALT / AST levels, the highest k value, and the highest percentage of in vitro ammonia clearance. The 10% HS flocculation medium produced results comparable to the ITS control condition and superior to the 5% HS and PG conditions. Based on LDH analysis, 10% HS produced the lowest total LDH amount per reactor during the flocculation process, but produced slightly better results compared to the ITS control condition during the encapsulation step. Similar behavior was observed in the ALT / AST test. 10% HS had the highest relative ammonia clearance after 72 hours among all groups.

[0175] Example 2. Optimization of cell density, fibroblast ratio, and culture time In this study, for example, the effects of total cell density, fibroblast to hepatocyte ratio, and duration of culture time were evaluated as key processing parameters for scale-up of the flocculation unit operation. Key outputs for optimization included aggregate ("seed") quantity, mean seed diameter, seed viability, and seed potency. In addition to these key outputs, additional outputs of ALT / AST / LDH enzyme secretion were also measured.

[0176] Cell density (total viable cells / culture medium volume, 3 × 10 5 ~1.5×10 6Experiments were performed to evaluate the following parameters: viable cells / mL), fibroblast to hepatocyte ratio (normal human dermal fibroblasts (NHDF):primary cultured human hepatocytes (PHH); 0.2-3.8), and incubation time (aggregation time 0-70 h). Table 3 shows the parameters that were tested.

[0177] [Table 3]

[0178] Important measurements included seed size, seed number, seed potency, seed viability, and NHDF:PHH ratio. Results are shown in Figures 33-37. In suspension, there are multiple modalities for scaling the seed generation process. Utilizing a design of experiments (DOE) approach allowed for the testing of multiple parameters in a single study. Here, a circumscribed central composite response surface design was employed. The design surface was repeated across several time points during aggregate formation in a vertical wheel bioreactor and consisted of nine points spanning reactor seeding density and fibroblast to hepatocyte ratio, with the central point repeated twice (group #5 and group #10). Model optimization was performed using proprietary modeling software. Several optima were observed across multiple experiments, and several patterns were observed across the three major response groups.

[0179] survival rate Higher hepatocyte-to-fibroblast ratios, lower densities, and less time were associated with the highest survival rates, suggesting a potential protective effect of fibroblasts on hepatocyte-specific viability measures, and possibly reflecting a tendency for fibroblasts to survive better than hepatocytes when measured by global viability measures.

[0180] Seed morphology The optimal curvature position of the morphology depends on the response evaluated. For seed size, intermediate values ​​in ratio, density, and time produced the largest size. For seed volume fraction, i.e., seed yield, ratio was not a significant factor, with high density and less time producing higher seed volume fractions. The tendency for longer times to produce higher yields is likely driven by the tendency for longer times to form superaggregates. In fact, for the formation of superaggregates, low ratios, intermediate density, and less time were best for minimal superaggregate formation. When normalized seed volume fractions are normalized by ALT, the only difference from the standard seed volume fractions is that the ALT normalized version emphasizes medium-high ratios, whereas the standard seed volume fractions are not affected by ratio.

[0181] efficacy Low ratios and high densities maximized the pure potency (ammonia decay constant) per reactor volume without any effect of time over t=22 or 46 h. Taking the ratio of potency per seed volume fraction did not change the optimum position, except for a slight preference for the later time point at t=46 h. Also, considering the ratio of potency per seed to superaggregate volume fraction, low ratios and late times are still preferred, except for an optimum being obtained at intermediate (but not high) densities. Taken together, the potency data suggest that lower ratios are associated with faster ammonia clearance, which may be due to a pure increase in hepatocytes per volume that is not apparently inhibited by metabolic constraints, overcoming the lower viability at lower ratios.

[0182] Overall, these results indicate that more hepatocytes result in higher potency. For producing seeds using the methods described herein, a NHDF:PHH ratio of 0.2 and a density of 1.5 M cells / mL were productive.

[0183] Example 3. Encapsulation of aggregates within a graft Satellite grafts were produced using seeds produced by the microwell or vertical wheel bioreactor. Seeds for groups 1, 3, and 4 were seeded in the microwells. Seeds for groups 2 and 5 were seeded in the VWB bioreactor. Grafts were transported as single satellite grafts per 15 mL conical tube in a portable incubator with a non-gas permeable cap. A total of 10 grafts were produced per group, plus 2 acellular FIBRYGA® graft controls (52 grafts total).

[0184] Seeds were harvested from the VWB or microwells using standard procedures and then centrifuged to form a master seed pellet. The master seed pellet was then resuspended in wash medium at a planned concentration of 18 M PHH / mL and further distributed into subbatches of two satellite grafts (volume 160 μL). Each subbatch was then rapidly mixed with thrombin and dispensed into casting molds. The grafts were then allowed to fully polymerize for 45 min at 37 °C in an incubator. Transport medium was added to the casting mold dish to hydrate the grafts during imaging. After imaging or 15 min after adding transport medium (whichever was longer), the grafts were manually transferred into transport tubes containing transport medium and then transported by courier for surgical implantation into the animals.

[0185] After transferring the master seed mixture from the 50 mL conical tube to subaliquot Eppendorf tubes, the original 50 mL conical tube was retained and the appearance of the seeds remaining on the surface of the conical tube was noted. Seeds produced by microwell aggregation appeared more irregular and clumpy compared to VWB-produced seeds, which exhibited a finer and more uniform appearance. Because VWB seeds are more easily suspended uniformly, distribution into subbatches is simplified. VWB seeds also appear to have a longer settling time in wash medium or fibrinogen, which is consistent with the smaller size of the seeds.

[0186] In vitro studies Two subbatches of 60 uL of seed fibrinogen suspension were produced and polymerized with thrombin (1:1 mixture similar to the in vivo grafts) to form surrografts. The surrografts were then transferred to gas permeable plates (separated by experimental condition) and treated with ammonium chloride. To assess ammonia clearance, samples were collected at 0 hours (i.e., 5 minutes after treatment), 5 hours, 24 hours, 48 ​​hours, and 72 hours. Two sets of surrograft controls were also evaluated; namely, Group 6: GMP housing, no cells, and Group 7: RUO housing, no cells. All groups showed similar ammonia clearance levels to previous data (Figures 38A and 38B). Group 3 had slightly lower ammonia clearance, which was most noticeable by 72 hours.

[0187] Suroseeds were prepared using the same seed fibrinogen suspension as the in vivo and ex vivo grafts, but were further diluted with fibrinogen (matching the original fibrinogen reagent) to a final concentration of 0.6M PHH / mL upon formation of the final drug product. Grafts were cultured in 24-well plates for 8 days, with medium changes and harvesting every 2 days. It was observed that the human albumin signal detected in grafts formed with human-derived housing reagents at early time points was higher than in grafts formed with bovine-derived housing reagents (Figure 39A). As part of the U1 Suroseed run, Group 6 (acellular FIBRYGA® grafts) was used in an attempt to evaluate the signal from the housing material alone. If we assume that the albumin signal is a linear combination of the albumin signal from the cells and the albumin signal from the housing reagents, the day-balanced albumin signal from Group 6 can be subtracted from the signal measured in Groups 2, 3, and 6 (as these are grafts formed with Fibryga). Preprocessing of this data was performed (Figure 39B). All grafts showed detectable U1 surrogate biomarker at all time points.

[0188] In vivo studies Grafts were implanted on day 0, then whole blood was collected and processed to plasma on days 4, 9, 14, 18, 23, and 28. Time points were chosen to accommodate a minimum of three blood draws within a two week period (Figure 40).

[0189] Biomarker Data - Albumin, Transferrin, and Correlations After implantation, drug products are integrated and PHH secrete human proteins into the bloodstream. Whole blood was collected, processed into plasma, and then frozen. Samples were then thawed and analyzed by ELISA targeting human albumin (Bethyl) or human transferrin (Abcam, ab187391). Values ​​for Group 2 (new product / treatment change) and controls (Groups 3-5) were statistically similar to baseline (Group 1) and sustained production all the way up to postoperative day 28 (Figure 41A). Plasma biomarker levels of albumin and transferrin were plotted in correlation plots (Figures 41B and 41C) and showed r-squared values ​​suggesting that these biomarkers are correlated with each other.

[0190] Histopathology review All grafts were explanted on postoperative day 28, stored in NBF fixative at room temperature for 24-30 hours, then transferred to 70% histology grade ethanol at room temperature, and then analyzed. Histological measurements were largely consistent with those of previous studies explanted at the same timeline (POD28-32), although some animals showed a higher degree of fibrosis and dystrophic calcification. Dystrophic calcification was rare in grafts of other 9M / mL dose density formulations, but a possible explanation is that grafts in GMP matrix have been shown to be softer and more brittle than the baseline formulation, which may lead to a higher incidence of graft folding and invagination within the graft bed, creating a macrostructure that leads to a less ideal metabolic environment. The degree of fibrosis appeared to be higher in GMP matrix compared to research grade matrix, and also in VWB aggregate seeds compared to microwell aggregate seeds. Furthermore, a pronounced degree of non-PHH cellularity in the graft area was observed, especially in grafts formed with GMP matrix.

[0191] 2D vessel analysis CK18 stained hepatocytes (Figure 42) and CD31 stained blood vessels (Figure 43) were used to analyze the vascular level of each group. Images were acquired using OlyVIA and hepatocyte aggregates were detected and counted using Cell Profiler (Figure 44). The aggregate areas were magnified (Figure 45) and a mask was generated defining the area containing the aggregates (Figure 46). This mask was then applied to the vascular image (Figure 47). CD31 staining was used in Angiotool as a marker of blood vessels within the masked area to evaluate the vascular network associated with the hepatocyte area. Evaluation metrics such as average vessel length, number of hepatocytes per square millimeter, number of blood vessels per square millimeter, and number of blood vessels per hepatocyte aggregate were calculated from the output of Angiotool and Cell Profiler analysis.

[0192] Analysis of average vessel length showed no significant differences between groups (Figure 48). This analysis was restricted to the area surrounding the hepatocytes and did not include the hydrogel headspace.

[0193] Analysis of hepatocyte aggregates per square millimeter showed significant differences between group 1 and groups 2 and 3, and neutral results with group 4 (Figure 49). This could be driven by histological sampling or even by seed administration per graft. However, seed pellet weight data showed that group 1 had the lowest seed pellet weight, 15% lower than the other groups, which is the opposite of what was expected. All other groups showed similar seed pellet weights.

[0194] Analysis of the number of blood vessels per square millimeter showed a trend but not a significant difference between Group 1 and Group 2 (p-value >0.05), and a significant difference between Group 2 and Group 4 (Figure 50). Groups 1 and 4 had similar blood vessel density, but variability in Group 1 is likely the cause of the lack of significance when compared to Group 2. The difference between Group 1 and Group 4 is that the ABM cells were grown in xeno-free medium and used in a format that thaws and aggregates. This means that the use of these cells does not directly translate to a decrease in blood vessel density. However, when these cells were used in VWB aggregates in the same hydrogel matrix (Group 5), blood vessel density was decreased. When these cells were used in F1 hydrogels rather than microwell aggregates, blood vessel density was decreased. Both VWB aggregates and F1 hydrogels contributed to the decrease in blood vessel density, which is consistent with the additive effect observed for the lowest blood vessel density in Group 2. This analysis of blood vessels per unit area was limited to the local area of ​​hepatocytes and did not explore the area of ​​the hydrogel.

[0195] Vascular density may depend on factors such as aggregate density, as well as fibroblast health and density. The fact that groups 1 and 4 were comparable rules out new sources and processing of fibroblasts as factors that decrease vascular density. The decrease in vascular density observed upon separation of VWB and F1 factors suggests that the retention of fibroblasts within the aggregates, and the nature of the hydrogel, may directly or indirectly affect vascular density.

[0196] Analysis of the number of blood vessels per hepatocyte aggregate showed no significant differences except between Group 4 and Group 5 (Figure 51). The difference between these groups is that Group 4 was a microwell aggregate and Group 5 was a vertical wheel aggregate. This indicated that the microwells had a higher number of blood vessels per aggregate. Groups 4 and 5 are in RUO fibrin. When comparing VWB and microwell aggregates in the background of GMP grade F1 hydrogel, the trend remained that VWB (Group 2) was slightly lower than microwell (Group 3), but the difference was not significant in the background of F1 hydrogel.

[0197] The number of blood vessels per hepatocyte aggregate is the most robust metric against potential sources of sampling bias, given that these data are derived from analysis of histological sections that do not necessarily represent the entire graft. All groups had approximately 3 blood vessels per hepatocyte aggregate.

[0198] Vascular analysis data was evaluated for correlation with secreted albumin levels. Both aggregate density (FIG. 52) and vessels per aggregate (FIG. 53) were evaluated, but no correlation was observed. Given that there is a linear correlation between albumin and transferrin, it would not have been expected that transferrin levels would have any correlation with these vascular measures.

[0199] Overall, these data indicate that grafts produced using aggregates produced by the vertical wheel bioreactor are vascularizable. Example 12. In vitro study to measure seed bed height in vertical wheel bioreactors Due to the need to control the seed bed height of the final graft for dose control, experiments were performed to evaluate seed bed height. Vertical wheel bioreactor (VWB) based seed bed heights showed a different trend than microwell based seed bed heights. We evaluated whether VWB seed bed heights generated from ROTEA™ washed NHDF, PHH and seeds were similar to non-ROTEA™ washed and / or microwell seed bed heights at dose densities of 3, 6 and 9 M / mL.

[0200] ROTEA™ washed NHDF and PHH after thawing and seeds after aggregation appear to produce slightly shorter seed bed heights on average compared to non-ROTEA™ washed seed bed heights at dose densities of 3, 6, and 9 M / mL (Figure 54). T-tests showed no significant differences between the two groups, indicating that ROTEA™ washing did not affect the formation of seed bed height. This also indicates that the debris and seeds washed away during the ROTEA™ protocol were not the seeds that formed the bulk of the seed bed height. This suggests that ROTEA™ removed cell debris and small seeds that did not settle quickly enough or debris observed in the fibrin headspace within the graft cross section. With the fibrin headspace more defined, the seed bed heights can be more clearly distinguished as seen in Figure 55.

[0201] In conclusion, these studies demonstrated that ROTEA™ washed cells and seeds can be utilized without having a significant effect on seed bed height. Example 13. Scaling of Bioreactor Production Experiments were performed to evaluate seed health as a function of VWB fill volume. NHDF and PHH were thawed from cryogenic storage, counted, and inoculated at the indicated concentrations in a ratio of 2 NHDF to 1 PHH in PBS MINI 3 x 0.1 L bioreactors or 1 x 0.5 L bioreactors. Four different fill volumes were tested: 1) 60 mL, 2) 75 mL (control), 3) 100 mL, and 4) 375 mL. Suspension cultures were allowed to run for 22 hours before harvesting. After 22 hours of aggregation, suspension cultures were harvested and samples were harvested and counted for analysis.

[0202] As shown in Figures 56A and 56B, ALT and AST levels remained constant among the different loading volumes (60 mL, 75 mL, 100 mL, and 375 mL), indicating that cell health was not adversely affected during scale-up of aggregation. Similarly, oxygen tension remained similar among the four loading volumes (Figure 57). Overall, these results suggest that the VWB system can be used to scale up bioreactor volumes for the production of aggregates.

[0203] Example 14. Maintenance of cell numbers during aggregation Experiments were performed to show that the method promotes aggregation and not proliferation, with the total number of cells not increasing substantially during aggregation.

[0204] PHH and NHDF populations were allowed to aggregate in suspension for 60 hours. Extracellular lactate dehydrogenase (LDH) and total lactate dehydrogenase were assessed at time points 0, 24, and 60 hours. Cell viability was also assessed (Table 4).

[0205] [Table 4]

[0206] As shown in Figures 58A and 58B, extracellular LDH in the medium accumulated over time. Total LDH remained constant over time, suggesting that the cells were not significantly proliferating. Taken together, these data suggest that more cells were dying over time and that the increase in extracellular LDH was not due to an increase in the number of cells.

[0207] Other embodiments While the invention has been described in conjunction with specific embodiments thereof, it will be understood that the invention is capable of further modifications, and that this application is intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention in general, including departures from the invention which have become known or are customarily practiced in the art to which the invention pertains, as applicable to the essential features described above, and in accordance with the scope of the appended claims.

[0208] Other embodiments are within the scope of the claims.

Claims

1. 1. A method for producing an aggregate of multiple cell populations, comprising: a first population of cells and a second population of cells; (a) agitating the liquid medium comprising the first population of cells and the second population of cells in a bioreactor for a duration sufficient to form aggregates comprising the first population of cells and the second population of cells, wherein the first population of cells and the second population of cells are in suspension within the liquid medium during the agitating step, thereby producing aggregates of the first population of cells and the second population of cells; (b) collecting the aggregates comprising the first population of cells and the second population of cells; The method comprising: (i) the first population of cells comprises fibroblasts; (ii) the second population of cells comprises parenchymal cells; and / or (iii) the plurality of cell populations further comprises one or more additional cell populations.

3. The fibroblasts (i) primary fibroblasts, induced pluripotent stem cell (iPSC)-derived fibroblasts, or embryonic stem cell (ESC)-derived fibroblasts; and / or (ii) The method of claim 2, wherein the fibroblasts are genetically engineered fibroblasts.

4. The method of claim 2 , wherein the parenchymal cells comprise hepatocytes or hepatocyte progenitor cells and / or pancreatic cells or pancreatic progenitor cells.

5. (I) The hepatocytes are (i) primary human hepatocytes, iPSC-derived hepatocytes, or ESC-derived hepatocytes, and / or (ii) genetically engineered hepatocytes; and / or (II) The pancreatic cells are (i) primary human pancreatic cells, iPSC-derived pancreatic cells, or ESC-derived pancreatic cells (ii) alpha cells, beta cells, gamma cells, delta cells, or epsilon cells, or a combination thereof; and / or 5. The method of claim 4, comprising (iii) genetically engineered pancreatic cells. (i) the first population of cells comprises fibroblasts and the second population of cells comprises hepatocytes; (ii) the first population of cells comprises fibroblasts and the second population of cells comprises hepatocyte progenitor cells; and / or 10. The method of claim 1, wherein (iii) the first population of cells comprises fibroblasts and the second population of cells comprises pancreatic beta cells. (i) a ratio of the first population of cells to the second population of cells is between 10:1 and 1:10; (ii) the ratio of the first population of cells to the second population of cells in the collected aggregates of step (b) is between 10:1 and 1:10; (iii) the first population of cells and / or the second population of cells are present at a density of 1×10 4 cells / mL to 1×10 8 cells / mL; (iv) the first population of cells and the second population of cells do not grow by more than 30% during the agitating step; and / or 10. The method of claim 1, wherein (v) the first population of cells and the second population of cells do not adhere to the bioreactor. (i) the first population of cells comprises fibroblasts, and the density of the fibroblasts is 1 x 10 ５ cells / mL ~ 1 x 10 ７ cells / mL or 6×10 5 cells / mL; 8. The method of claim 7, wherein (ii) the second population of cells comprises hepatocytes, and the density of the hepatocytes is between 1 x 10 cells / mL and 1 x 10 cells / mL or 3 x 10 cells / mL.

9. The bioreactor is (i) having a capacity of 0.1 L to 500 L; (ii) a stirred tank bioreactor or a vertical wheel bioreactor; (iii) further comprising a biotreatment controller that controls one or more of pH, temperature, and dissolved oxygen concentration; and / or (vi) The method of claim 1, wherein the incubation is carried out at a temperature of 35°C to 39°C. (i) at least 80% of the aggregates have an average diameter of ±10% of each other; and / or (ii) The method of claim 1, wherein the aggregates are spheroids.

11. The stirring step (i) comprises a duration of between 1 hour and 72 hours, up to 18 hours, or up to 24 hours; and / or 10. The method of claim 1, comprising (ii) rotating the bioreactor at a speed of between 10 RPM and 50 RPM.

12. The medium (i) has a viscosity of 0.9 cP to 1.4 cP; (ii) containing 1 to 20 μg / mL of recombinant human insulin, 1 to 10 μg / mL of human transferrin, and 1×10 −3 to 1×10 −2 μg / mL of selenite; and / or The method of claim 1 , comprising laminin, collagen, elastin, or fibronectin.

13. The medium (i) 1 to 20 μM of a Rho-associated protein kinase (ROCK) inhibitor; (ii) human serum; (iii) platelet lysate; (iv) 4,000 mg / L to 5,000 mg / L glucose; (v) 10 ng / mL to 100 ng / mL of glucagon; (vi) fibrinogen, and / or (vii) 10 ng / mL to 100 ng / mL of dexamethasone. (i) the ROCK inhibitor is Y27632; (ii) the medium contains 0.1% to 20% (v / v) human serum, and / or (iii) The method of claim 13, wherein the medium comprises 0.1% to 10% (v / v) platelet lysate.

15. The method (i) producing an aggregate density of 500 aggregates / mL to 10,000 aggregates / mL; (ii) producing an average aggregate mean diameter of between 50 μm and 200 μm; and / or (iii) producing an average total volume of aggregates between 200 μL and 50 mL.

16. Each of the aggregates is at least 10 ３ 10. The method of claim 1, wherein the cells comprise at least 100 cells and / or each have an average diameter of at least 50 μm. (i) washing the cells prior to step (a); (ii) washing, purifying, and / or concentrating the collected aggregates of step (b); (iii) formulating the collected aggregates of step (b) in a storage buffer; and / or (iv) encapsulating the collected aggregates of step (b) within a biocompatible scaffold (c). The method of claim 1 further comprising:

18. The method described in claim 17, wherein the method includes purifying the collected aggregates of step (b), and the collected aggregates are purified by centrifugation or acoustic separation.

19. 1. A method of encapsulating an aggregate of multiple cell populations, the aggregate comprising a first population of cells and a second population of cells, the method comprising: (a) agitating the liquid medium comprising the first population of cells and the second population of cells in a bioreactor for a duration sufficient to form aggregates comprising the first population of cells and the second population of cells, wherein the first population of cells and the second population of cells are in suspension within the liquid medium during the agitating step, thereby producing aggregates of the first population of cells and the second population of cells; (b) collecting the aggregates of step (a); (c) encapsulating the aggregate of step (b) within a biocompatible scaffold.

20. 20. The method of claim 17 or 19, wherein encapsulating the aggregate comprises providing a polymerization agent or a cross-linking reagent to polymerize or cross-link the biocompatible scaffold, thereby encapsulating the aggregate.

21. 20. The method of claim 17 or 19, wherein the biocompatible scaffold comprises fibrinogen and / or the biocompatible scaffold further comprises a reinforcement agent.

22. 21. The method of claim 20, wherein the polymerization agent comprises thrombin.

23. 22. The method of claim 21, wherein the reinforcing agent comprises collagen, poly(ethylene glycol), polyvinylidene acetate (PVDA), polyvinylidene fluoride (PVDF), poly(lactic-co-glycolic acid) (PLGA), or poly(L-lactic acid) (PLLA).

24. (i) washing the cells prior to step (a); (ii) washing, purifying, and / or concentrating the collected aggregates of step (b) prior to encapsulation; and / or 20. The method of claim 19, further comprising (iii) formulating the collected aggregates of step (b) in a storage buffer prior to encapsulation.

25. 25. The method of claim 24, wherein at least 80% of the aggregates have an average diameter of each other ±10% after purification.

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