The Effects of Metabolic Substrate Availability on Human Adipose-Derived Stem Cell Spheroid Survival

Abstract

Human adipose-derived stem cells (hADSCs) spheroids have displayed remarkable potential for treating ischemic injury. However, low nutrient (i.e., glucose and oxygen) availability in ischemic environments results in limited tissue viability posttransplantation. To develop an understanding of the effects of nutrient availability on spheroid survival, we utilized both in vitro and computational models to examine the limiting factors in metabolic supply for avascular microtissues, revealing the critical role of glucose to improve hADSC spheroid survival in ischemic conditions. These results may impact future strategies for improving hADSC transplantation efficacy through codelivery of metabolic substrates.

Keywords: 3D tissue culture; adipose-derived stem cell; ischemia; mathematical modeling; nutrient diffusion; spheroid.

FIG. 1.

hADSC spheroids provide an ideal model to test the effects of nutrient availability on viability. (A) Representative images of hADSC spheroids of four radii (115, 135, 175, and 215 μm). (B) Fabrication techniques produced hADSC spheroids of consistent and significantly different size (x-axis nominal radii, y-axis measured radii). (C) General nutrient consumption surface plot representing how nutrient (i.e., glucose/oxygen) concentrations decrease contiguously from the spheroid surface to the center. Low nutrient levels at the center may not be sufficient to meet hADSC metabolic demands and result in an apoptotic core. Color images available online at www.liebertpub.com/tea

FIG. 2.

hADSC viability is dependent upon spheroid size and availability of metabolic substrates. (A) Representative immunofluorescence images showing TUNEL staining of hADSC spheroids after 72 h of exposure to each of the defined testing conditions. Four culture conditions were chosen to assess the effects of metabolic availability on hADSC spheroids. These conditions include Control (+Oxy/+Glu, 21% oxygen; 5.5 mM glucose), Ischemia (−Oxy/−Glu, 1% oxygen; 0.55 mM glucose), Ischemia + Glucose (−Oxy/+Glu, 1% Oxygen; 5.5 mM glucose), and Ischemia + Oxygen (+Oxy/−Glu, 21% oxygen; 0.55 mM glucose). Scale bar = 100 μm. (B) Quantification of spheroid viability after 72 h of exposure under each condition for each size group. For each culturing condition, n = 8 spheroids. ^#,##Significant difference between ischemic conditions across spheroids size groups (p < 0.05). *Significant difference between culture conditions for each spheroid size group (p < 0.05). Color images available online at www.liebertpub.com/tea

FIG. 3.

Glucose concentration and consumption rates are dependent upon spheroid size and culture conditions. (A) Plots showing modeled glucose concentrations for different-sized spheroids under control (+Oxy/+Glu) and ischemic (−Oxy/−Glu) conditions. (B) Plots showing modeled glucose concentrations for different-sized spheroids under ischemic + glucose (−Oxy/+Glu) and ischemic + oxygen (+Oxy/−Glu) conditions. Glucose concentration for Control and −Oxy/+Glu are identical (same trend for Ischemia and +Oxy/−Glu). (C–F) Plots showing modeled glucose consumption rates under “High Glucose” (Control, −Oxy/+Glu) and “Low Glucose” (Ischemic, +Oxy/−Glu) conditions for spheroid radii of (C) 115 μm, (D) 135 μm, (E) 175 μm, and (F) 215 μm. Color images available online at www.liebertpub.com/tea

FIG. 4.

Increased glucose availability positively correlates with improved hADSC viability. (A) Plot shows the relationship between glucose consumption rate with respect to spheroid volume at the High- and Low-glucose culture conditions. The dotted line defines the glucose consumption rate threshold, at which functional glucose consumption is depleted. (B) Plot illustrating that increased glucose availability positively correlates with increased spheroid viability (p < 0.001). Color images available online at www.liebertpub.com/tea

FIG. 5.

Lactate concentration and accumulation is dependent upon spheroid size and culture conditions. (A, B) Plots showing lactate concentrations recorded periodically over a 72 h time period for spheroids of 115 μm (A) and 175 μm (B) radii under control (+Oxy/+Glu), ischemic + glucose (−Oxy/+Glu), ischemic + oxygen (+Oxy/−Glu), and ischemic (−Oxy/−Glu) culturing conditions. For each culture condition and observation period, n = 3. (C, D) Plots showing modeled lactate concentrations under control (+Oxy/+Glu), ischemic + glucose (−Oxy/+Glu), ischemic + oxygen (+Oxy/−Glu), and ischemic (−Oxy/−Glu) culturing conditions for spheroid radii of (C) 115 μm and (D) 175 μm. Color images available online at www.liebertpub.com/tea

FIG. 6.

Modeling ATP production in hADSC spheroids. (A) Total ATP production profiles for spheroids of r = 135 μm under four culture conditions. (B) Distributions of glycolytic to total ATP ratio in spheroids of r = 135 μm under four nutrient conditions. The curve for the ischemic and +Oxy/−Glu groups end at the radial position, at which ATP production ceased due to glucose depletion. (C) Investigation of preferential hADSC dependence on anaerobic glycolysis through mitochondrial inhibition on spheroid (∼135 μm) viability under different culture conditions. ATP, adenosine triphosphate. Color images available online at www.liebertpub.com/tea

FIG. 7.

Increased glucose availability sustains cell viability over time in larger spheroids. (A–D) Quantification of cell viability when comparing ischemic (−Oxy/−Glu) and ischemia + glucose (−Oxy/+Glu) culture conditions at 0, 3, 6, 24, 48, and 72 h for (A) 115 μm, (B) 135 μm, (C) 175 μm, and (D) 215 μm spheroids. For each culture condition and time point, n = 8 spheroids for each spheroid size group and time point. *Significant difference between culture conditions at each time point between spheroid size groups (p < 0.05). Color images available online at www.liebertpub.com/tea