cryopreserved phh lots  (Thermo Fisher)

Journal: Gene Expression

Article Title: Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models

doi: 10.3727/105221620X15868728381608

Figure Lengend Snippet: Effects of PHH homotypic interactions within microtissues on liver functions. Collagen-based microtissues containing increasing densities of PHHs in a pH-neutralized collagen (1.25e6 and 2.5e6 cells/ml) solution were fabricated using the droplet microfluidic device shown in Figure 1. Representative phase-contrast images of PHH microtissues with a density of (a) 1.25e6 cells/ml and (b) 2.5e6 cells/ml placed within agarose microwells cast within 24-well plates. Both 1.25e6 and 2.5e6 cells/ml densities produced reproducible microtissues with high yields that fit within the 300 μm × 300 μm agarose microwells. (c) Albumin production and CYP3A4 enzyme activity for PHH microtissues fabricated using a cell density of 1.25e6 and 2.5e6 cells/ml. Microtissues with 3.75e6 and 5e6 cells/ml densities were also fabricated, but these densities did not produce homogenously sized microtissues (not shown). Statistical significance is displayed relative to 1.25e6 cells/ml at the same time point. ***p ≤ 0.001.

Article Snippet: Cryopreserved PHHs (lot HUM4055A, 54-year-old female Caucasian, and HUM4192, 16-year-old female Asian; Lonza, Walkersville, MD, USA) were thawed, counted, and viability (>85%) was assessed as previously described 21 .

Techniques: Produced, Activity Assay

Journal: Gene Expression

Article Title: Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models

doi: 10.3727/105221620X15868728381608

Figure Lengend Snippet: Comparison of PHH microtissues with conventional self-assembled spheroids and macrogels. Schematics and phase-contrast images for the tested culture models: (a) microtissue containing collagen and PHHs formed using the droplet microfluidic device of Fig. 1; (b) self-assembled PHH spheroids without any collagen scaffolding; (c) macrogels in which the PHH + collagen mixture is dispensed directly into multiwell plates without being subjected to a droplet microfluidic device. (d) Albumin production from PHH-only microtissues, spheroids, and macrogels. Statistical significance is displayed for microtissues relative to spheroids (***p ≤ 0.001, and ****p ≤ 0.0001) and for microtissues relative to macrogels (++++p ≤ 0.0001). Activities of different CYP450 isoenzymes (e) CYP3A4, (f) CYP2A6, (g) CYP2C9, and (h) CYP1A2 in PHH-only microtissues, spheroids, and macrogels. Statistical significance is displayed relative to microtissues at the same time point. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. Arrows indicate undetectable levels for the indicated function and indicated culture model.

Article Snippet: Cryopreserved PHHs (lot HUM4055A, 54-year-old female Caucasian, and HUM4192, 16-year-old female Asian; Lonza, Walkersville, MD, USA) were thawed, counted, and viability (>85%) was assessed as previously described 21 .

Techniques: Comparison, Scaffolding

Journal: Gene Expression

Article Title: Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models

doi: 10.3727/105221620X15868728381608

Figure Lengend Snippet: Effects of 3T3-J2 fibroblasts and primary human hepatic stellate cells (HSCs) on PHH microtissues. Phase-contrast images for tested culture models: (a) Only PHHs encapsulated within collagen microtissues; (b) PHHs and 3T3-J2 fibroblasts encapsulated together within the collagen microtissue [coencapsulated (3T3-J2)]; (c) 3T3-J2 fibroblasts seeded/coated onto the surface of the polymerized collagen-based PHH microtissues [coated (3T3-J2)]; (d) primary HSCs seeded/coated onto the surface of the polymerized collagen-based microtissues [coated (HSC)]. The smaller size of the microtissues with coculture indicate compaction of the microtissues by the fibroblasts. (e) Albumin secretions from PHH-only, PHH/3T3-J2-coencapsulated, PHH/3T3-J2-coated, and PHH/HSC-coated microtissues. Activities of different CYP450 isoenzymes, (f) CYP3A4, (g) CYP2A6, (h) CYP2C9, (i) CYP1A2 in PHH-only, PHH/3T3-J2-coencapsulated, PHH/3T3-J2-coated, and PHH/HSC-coated microtissues. Statistical significance is displayed relative to PHH-only microtissues (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Arrow indicates undetectable level for the indicated function and indicated culture model.

Article Snippet: Cryopreserved PHHs (lot HUM4055A, 54-year-old female Caucasian, and HUM4192, 16-year-old female Asian; Lonza, Walkersville, MD, USA) were thawed, counted, and viability (>85%) was assessed as previously described 21 .

Techniques:

Journal: Gene Expression

Article Title: Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models

doi: 10.3727/105221620X15868728381608

Figure Lengend Snippet: Projected surface area distributions of 3D human liver models. Collagen-based microtissues containing encapsulated PHHs were fabricated using the droplet microfluidic device shown in Figure 1, while self-assembled spheroids were created as shown in Figure 3. Collagen-based microtissues containing encapsulated PHHs and 3T3-J2 fibroblasts were fabricated to create coencapsulated microtissue. 3T3-J2 fibroblasts were coated onto the PHH microtissues or spheroids to create coated microtissue and coated spheroid, respectively. Spheroid and Microtissue indicate models with only PHHs. After 21 days of culture, 375 individual constructs for each model were fixed and analyzed via phase-contrast microscopy for projected surface area determinations. Shown here are the histograms of the projected surface areas for the five culture/coculture models.

Article Snippet: Cryopreserved PHHs (lot HUM4055A, 54-year-old female Caucasian, and HUM4192, 16-year-old female Asian; Lonza, Walkersville, MD, USA) were thawed, counted, and viability (>85%) was assessed as previously described 21 .

Techniques: Construct, Microscopy

Journal: Gene Expression

Article Title: Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models

doi: 10.3727/105221620X15868728381608

Figure Lengend Snippet: Effects of paracrine-only interactions between 3T3-J2 fibroblasts and PHH microtissues. Schematics for tested culture models: (a) Only PHHs encapsulated within collagen microtissues (control model); (b) PHH microtissues with 3T3-J2 fibroblasts seeded into a Transwell insert (3T3-J2 Transwell); (c) 3T3-J2 fibroblasts coated onto the surface of the polymerized collagen-based PHH microtissues (3T3-J2 coated, control model). Activities of different CYP450 isoenzymes, (d) CYP3A4, (e) CYP2A6, (f) CYP2C9, (g) CYP1A2 in PHH-only, 3T3-J2 Transwell, and 3T3-J2-coated microtissues. Statistical significance is displayed for 3T3-J2 Transwell microtissues relative to PHH-only microtissues (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Statistical significance is displayed for 3T3-J2-coated microtissues relative to 3T3-J2 Transwell microtissues (+p ≤ 0.05, ++p ≤ 0.01, +++p ≤ 0.001, ++++p ≤ 0.0001).

Article Snippet: Cryopreserved PHHs (lot HUM4055A, 54-year-old female Caucasian, and HUM4192, 16-year-old female Asian; Lonza, Walkersville, MD, USA) were thawed, counted, and viability (>85%) was assessed as previously described 21 .

Techniques: Control

Journal: Gene Expression

Article Title: Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models

doi: 10.3727/105221620X15868728381608

Figure Lengend Snippet: Long-term gene expression in 3T3-J2-coated and HSC-coated microtissues. 3T3-J2 fibroblasts or primary human HSCs were coated onto the surface of the polymerized collagen-based PHH microtissues and cultured for ∼6 weeks. Quantitative gene expression of albumin, ornithine transcarbamylase (OTC), hepatic transcription factors (CEBPa and HNF4a), CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4), canalicular transporter (MRP2), basolateral transporter (SLCO1B1), and nuclear receptors (AHR, CAR, PXR, and RXR) in 3T3-J2-coated microtissues (a) and HSC-coated microtissues (b) at 2, 4, and 6 weeks as compared to freshly thawed PHHs (line 20) that were immediately lysed in suspension for RNA (i.e., expression levels in the coated microtissues at the 20 line are near identical to the levels in freshly thawed PHHs). Arrows indicate detectable levels at the 20 line. For expression levels denoted ND (not detectable), amplification was not observed for the given gene within 40 cycles of quantitative polymerase chain reaction (qPCR). Statistical significance for gene expression levels is displayed for 3T3-J2 coated and HSC coated at 2, 4, and 6 weeks relative to freshly thawed PHHs (*p ≤ 0.05, +p ≤ 0.01, ‡p ≤ 0.001, #p ≤ 0.0001).

Article Snippet: Cryopreserved PHHs (lot HUM4055A, 54-year-old female Caucasian, and HUM4192, 16-year-old female Asian; Lonza, Walkersville, MD, USA) were thawed, counted, and viability (>85%) was assessed as previously described 21 .

Techniques: Gene Expression, Cell Culture, Suspension, Expressing, Amplification, Real-time Polymerase Chain Reaction

Journal: Gene Expression

Article Title: Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models

doi: 10.3727/105221620X15868728381608

Figure Lengend Snippet: Drug-mediated CYP induction and drug-induced hepatotoxicity in 3T3-J2-coated microtissues. Coated microtissues were allowed 9 days while 2D PHH monocultures were allowed 1 day to functionally stabilize, and then both culture models (same PHH donor/lot) were treated with rifampin or omeprazole for 4 days with fresh drug added to the culture medium at the 2-day medium exchange. CYP activities were assessed at the end of the 4-day incubation period. (a) CYP3A4 and CYP2C9 activities in 2D monocultures and coated microtissues treated with rifampin, and CYP1A2 activity in 2D monocultures and coated microtissues treated with omeprazole. Enzyme activities in the drug-treated cultures were normalized to enzyme activity in cultures treated with DMSO alone. Statistical significance directly above each bar is displayed relative to DMSO-treated controls (**p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). Statistical significance above each line is displayed for coated microtissue induction relative to 2D monoculture induction (**p ≤ 0.01). Additionally, coated microtissues were allowed ∼9 days to functionally stabilize and then treated with rosiglitazone or troglitazone at two concentrations for each drug, 30× and 60× Cmax (Cmax = maximum drug concentration measured in human plasma; Cmax for troglitazone = 2.82 μg/ml and Cmax for rosiglitazone = 0.373 μg/ml), for a total of 8 days with fresh drug added to culture medium at the 4-day medium exchange. (b) Albumin secretion, specific to PHHs (top graph), and overall coculture viability (bottom graph, PrestoBlue™ conversion to the metabolite, resazurin) in coated microtissues treated with rosiglitazone. Data in drug-treated coated microtissues were normalized to the corresponding data in DMSO-only treated cultures. (c) Similar graphs as (a) except coated microtissues were treated with troglitazone. For drug toxicity data, statistical significance is relative to DMSO-treated control cultures. *p ≤ 0.05, ***p ≤ 0.001, and ****p ≤ 0.0001.

Article Snippet: Cryopreserved PHHs (lot HUM4055A, 54-year-old female Caucasian, and HUM4192, 16-year-old female Asian; Lonza, Walkersville, MD, USA) were thawed, counted, and viability (>85%) was assessed as previously described 21 .

Techniques: Incubation, Activity Assay, Concentration Assay, Clinical Proteomics, Control