Infected Cell Cultures

Abstract

3D cell cultures contain hepatic cells and are infected by a pathogen. Such cell cultures are prepared by, for example, inoculating a single-cell suspension containing hepatic cells expanded in a 2D culture in an agitation-based culture system. Next, the resulting cell culture is agitated at a given agitation rate. Then, the resulting 3D cell culture containing cell aggregates is incubated with a pathogen.

Claims

1: A 3D cell culture, comprising: cell aggregates, which contain hepatic cells, wherein the cell aggregates are infected by a pathogen.

2: The 3D cell culture according to claim 1, wherein the pathogen is a parasite.

3: The 3D cell culture according to claim 1, wherein the 3D cell culture is a mono-culture or a co-culture.

4: The 3D cell culture according to claim 1, wherein the hepatic cells are selected from at least one cell source selected from the group consisting of primary human hepatocytes, murine hepatocytes, primate hepatocytes, cell lines hepatocyte-like cells derived from pluripotent stem cells, and hepatocyte-like cells derived from multipotent stem cells.

5: The 3D cell culture according to claim 1, wherein the cell aggregates have an average diameter in the range of 50 μm to 200 μm.

6: The 3D cell culture according to claim 2, wherein the parasite is from the genus Plasmodium.

7. The 3D cell culture according to claim 1, wherein the pathogen is a reporter strain.

8: The 3D cell culture according to claim 1, which contains a culture medium.

9: The 3D cell culture according to claim 1, further comprising a soluble extracellular matrix.

10: A multi-well plate containing the 3D cell culture according to claim 1.

11: A method for the production of a 3D cell culture containing hepatic cells, said method comprising: (a) inoculating a single-cell suspension, containing hepatic cells expanded in 2D culture, in an agitation-based culture system; (b) agitating the resulting cell culture at an agitation rate of 40 to 110 rpm; and/or (c) incubating the resulting 3D cell culture containing cell aggregates with a pathogen.

12: The method for the production of a 3D cell culture according to claim 11, wherein the incubation is performed under static conditions, wherein the 3D cell culture containing the cell aggregates, together with the pathogen, is exposed to centrifugation at up to 1800×g, or wherein the incubation is performed under dynamic conditions, wherein the cell culture volume is reduced, and the 3D cell culture is exposed to agitation.

13: The 3D cell culture containing hepatic cells obtainable with the method according to claim 11.

14: A screening method, comprising: (a) incubating a 3D cell culture containing hepatic cells with a compound, wherein the 3D cell culture is the cell culture according to claim 1; and (b) monitoring of pathogen invasion, compound clearance and/or development of host cells.

15: A method, comprising: contacting a compound with the 3D cell culture according to claim 1, and determining a cytotoxic effect and/or metabolic properties of the compound contacted with the 3D cell culture and/or an effect of the compound contacted with the 3D cell culture on the pathogen.

16: A vaccine, comprising: the 3D cell culture according to claim 1.

17: A screening assay for an anti-parasitic drug and/or a vaccine, comprising: a 3D cell culture containing hepatic cells, wherein the 3D cell culture is according to claim 1, and a compound.

18: A kit for the screening for a drug and/or a vaccine, comprising: the 3D cell culture according to claim 1.

19: The 3D cell culture according to claim 4, wherein the at least one cell source is at least one cell line selected from the group consisting of HC-04, HepG2, HepaRG, and Huh7.

20: The 3D cell culture according to claim 9, wherein the soluble extracellular matrix comprises at least one material selected from the group consisting of laminin, fibronectin, collagen and a biocompatible biomaterial.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0069] FIG. 1—Example of a possible compound incubation regime to be used on drug screening for anti-Plasmodium agents. (A) Incubation of the drug from 1 hour before the addition of the pathogens up to 2 or 7 days later; (B) Incubation of the drug for 1 hour before the addition of Plasmodium sporozoites; (C) Incubation of the drug upon the addition of Plasmodium sporozoites for 2 hours; (D) Incubation of the drug 2 hours from the time of addition of Plasmodium sporozoites up to 2 or 7 days.

[0070] FIG. 2—Characterization of HepG2 spheroids during 3D culture. (A) Phase contrast and fluorescence microscopy images of live/dead assay (Live cells, Fluorescein-diacetate; Dead cells, Topro-3) in the first and second weeks of culture (day 4 and 9, respectively). Scale bars: 50 μm. (B) Spheroid diameter in the first and second weeks of culture (Days 4 and 9, respectively). Results are presented as mean±S.D of two independent experiments. (C) Analysis of gene expression levels of 2D cultures and 3D cultures over 15 days of culture for the metabolic genes CYP3A4, CYP2D6 and CYP1A2. Results are presented as mean±SEM of two or three independent experiments.

[0071] FIG. 3—Characterization of HepaRG spheroids during 3D culture. (A) Phase contrast and fluorescence microscopy images of live/dead assay (Live cells, Fluorescein-diacetate; Dead cells, Topro-3) during the first and second weeks of culture (Day 4 and 9, respectively). Scale bars: 50 μm. (B) Spheroid diameter in the first and second weeks of culture (Days 4 and 9, respectively). Results are presented as mean±S.D of two independent experiments.

[0072] FIG. 4—Optimization of the aggregation of HC-04 cells. Aggregation was induced by culturing the cells for 3 days in medium with 10% to 20% (v/v) FBS. Contrast phase microscopy images representative of HC-04 spheroids from the 2 culture conditions by day 6 of culture. Scale bars: 50 μm.

[0073] FIG. 5—Characterization of HC-04 spheroids during 3D culture. (A) Phase contrast and fluorescence microscopy images of live/dead assay (Live cells, Fluorescein-diacetate; Dead cells, Topro-3) in the second week of culture (Day 9). Scale bars: 50 μm. (B) Spheroid diameter in the first and second weeks of culture (Days 4 and 9, respectively). Results are presented as mean±S.D. of three independent experiments. (C) Analysis of gene expression levels of 2D cultures at day 3 and 3D cultures over 15 days of culture for the metabolic genes CYP3A4, CYP1A2 and CYP2D6. Results are presented as mean±SEM of two or three independent experiments.

[0074] FIG. 6—Phenotypic characterization of HC-04 spheroids. Detection of: (A) E-cadherin; (B) F-actin; (C) Albumin; (D) Hepatocyte nuclear factor 4 alpha (HNF4α); (E) CYP3a4; (F) CD81. Images from fluorescence microscopy of 10 μm thick cryosections of spheroids from day 9. Scale bars: 50 μm.

[0075] FIG. 7—Characterization of PHH spheroids during 3D culture. (A) Phase contrast and fluorescence microscopy images of showing dead cells (Dead cells, Topro-3) at day 3 and 6 of culture, respectively. Scale bars: 100 μm.

[0076] FIG. 8—Characterization of of heterotypic spheroid cultures. (A) Phase contrast microscopy image of PHH:HepaRG co-culture at day 3 of culture. Scale bars: 50 μm. (B) Phase contrast microscopy images of HC-04:HepaRG co-culture at day 4 of culture. Scale bars: 50 μm.

[0077] FIG. 9—Characterization of P. berghei infection of 3D cultures in dynamic conditions. (A) Phase contrast and fluorescence microscopy images of live/dead assay (Live cells, Fluorescein-diacetate; Dead cells, Topro-3) 48 hours after infection. Scale bars: 100 μm. (B) Infection rate of 3D cultures infected in static and dynamic conditions, expressed as percentage relative to static infection. Luciferase activity was normalized by μg of DNA. Results are the mean±S.D of 4 technical replicates from a single experiment.

[0078] FIG. 10—Optimization of HepG2 spheroid culture conditions for infection. Fluorescence microscopy images of viable cells (Fluorescein-diacetate) of HepG2 spheroids after centrifugation at 500, 1000 and 1800 xg, the latter with slow acceleration and braking. Scale bars: 100 μm.

[0079] FIG. 11—Culture of HepG2 spheroids in 96 well plates. Fluorescence microscopy images of viable cells (Fluorescein-diacetate) of HepG2 spheroids centrifuged at 1800×g for 5 minutes with medium acceleration and brake, and maintained for an additional 48h in the 96-well plate. Scale bars: 100 μm.

[0080] FIG. 12—Optimization of cell-to-sporozoite ratio and mode of contact. (A) Luciferase activity in relative luminescence units (RLU) for centrifuged (black) and non-centrifuged (gray) conditions. Results are the mean of 5 technical replicates±S.D from a single experiment. (B) Luciferase activity of infected HepG2 spheroids relative to the infection of HepG2 2D cultures. Results are the average of at least 3 independent biological experiments±SEM.

[0081] FIG. 13—Optimization of cell density at infection. Contrast phase microscopy images of first and second week HepG2 spheroids distribution in a 96-well plate. Scale bars: 100 μm.

[0082] FIG. 14—Optimization of Plasmodium infection of HepG2 spheroids. Infection rate of HepG2 spheroids by Pb-Luc (A) and Pb-GFP (B) relative to HepG2 cells cultured in 2D infected at 1:1 cell-to-sporozoite ratio. Results are represented as the mean±SEM of at least 5 independent experiments. (C) Development of Pb-GFP parasites in HepG2 spheroids. Results of GFP intensity normalized to those obtained for HepG2 cells cultured in 2D. Results are presented as mean±EM of at least 5 independent experiments. * indicate significant differences by a t-student test (**p<0.01, *p<0.05).

[0083] FIG. 15—Plasmodium infection of HC-04 spheroids. Infection rate of Pb-Luc (A) and Pb-GFP (B) for HC-04 spheroids and 2D cell cultures normalized for HepG2. Results represented as the mean±SEM of at least 3 independent experiments. (C) Development of Pb-GFP parasites in HC-04 spheroids. Results of GFP intensity normalized to those obtained for HepG2 cultured in 2D. Results are presented as mean±SEM of at least 3 independent experiments. * indicate significant differences by a t-Student test (*p<0.05).

[0084] FIG. 16—Quantification of Plasmodium infection in HC-04 spheroids. (A) Percentage of infected spheroids at 2.5 and 5×10.sup.4 cell/well in a 1:2 cell-to-sporozoite ratio. Results are mean±S.D of two independent experiments. (B) Number of cells infected per spheroid, infected in 2.5 and 5×10.sup.4 cell/well in a 1:2 cell-to-sporozoite ratio. Results are mean±S.D of two independent experiments. (C) Fluorescence microscopy image representative of Pb-GFP infection. Scale bar: 100 μm.

[0085] FIG. 17—Characterization of Plasmodium development throughout infection. (A) Pb-GFP development over time (24 to 60 h post-infection) in 2D and 3D cultures of HepG2 and HC-04, determined by quantification of GFP intensity. Results are mean±S.D of three independent experiments (B) Monitoring of sporozoites growth within infected cells at 24 h, 36 h and 48 h post-infection in HC-04 spheroid and 2D cultures. Arrows indicate cells with developing parasites. (C) Detection of the UIS4 parasitophorous vacuole protein at 48 h post-infection using a specific α-UIS-4 antibody. 3D images are projections of 4.2 μm z-stacks. Scale bars: 50 μm.

[0086] FIG. 18—In vivo infectivity of merosomes from 3D cultures of HC-04 and 2D cultures of HepG2, determined by quantification of infected red blood cells (RBC). Results are mean±S.D of one experiment including at least four mice per condition.

[0087] FIG. 19—Analysis of drug activity in Pb infected-HC-04 spheroids. Dose-response curve of 3D cultures treated with ATQ. Results are expressed as percentage of infection normalized to the non-treated control. Results are represented as the mean of up to two independent experiments.

[0088] FIG. 20—Schematic representation of the preparation of an infected 3D culture according to the present invention and use of the same in a high-throughput screening for anti-malaria drugs.

EXAMPLES

[0089] Unless otherwise specified, all starting materials are obtained from commercial suppliers and used without further purifications. Unless otherwise specified, all temperatures are expressed in ° C. and all reactions are conducted at RT.

Abbreviations

ATQ—Atovaquone

DMEM—Dulbecco's Modified Eagle's Medium

DMSO—Dimethyl Sulfoxide

[0090] ECM—Extracellular matrix
FBS—Fetal bovine serum

F12—Ham's F-12 Nutrient Mixture

[0091] GFP—Green fluorescent protein
HNF4α—Hepatocyte nuclear factor 4 alpha
P. berghei—Plasmodium berghei
P. cynomolgi—Plasmodium cynomolgi
P. falciparum—Plasmodium falciparum
P. malariae—Plasmodium malariae
P. ovale—Plasmodium ovale
P. vivax—Plasmodium vivax
Pb-GFP—Plasmodium berghei constitutively expressing GFP
Pb-Luc—Plasmodium berghei, constitutively expressing luciferase
RLU—Relative luminescence units
rpm—Rotations per minute

S.D—Standard Deviation

[0092] STB—Stirred-tank bioreactors
xg—x times gravity

[0093] The invention will be illustrated (but not limited), by reference to the specific embodiments described in the following examples.

I. Growing and Characterization of the 3D Cultures

Example 1a: Establishment of 3D Culture of HepG2 Cells

[0094] HepG2 spheroids were generated in stirred-tank systems. The culture conditions used for HepG2 spheroids are summarized in Table 1.

[0095] HepG2 cells formed spheroids with high cell viability (FIG. 2). By day 4, HepG2 spheroids were compact, with an average diameter of 63±14 μm (FIG. 2, day 4). Although spheroids presented higher diameter heterogeneity by the second week of culture (FIG. 2, day 9), they are more compact than in the first week, with an average diameter of 104±32 μm (FIG. 2 B). Analysis of basal gene expression of CYP3A4, 2D6 and 1A2 over time indicated that there are no major differences in gene expression over the culture period, showing the metabolism is stable in 3D culture over time (FIG. 2 C).

TABLE-US-00001 TABLE 1 Culture conditions for the establishment of 3D culture of HepG2 cells. Cell inoculum 0.3 × 10.sup.6 cell/mL concentration Culture medium DMEM + 10% (v/v) FBS + 1% (v/v) PenStrep Agitation rate 40-100 rpm Feeding regimen 50% (v/v), every three days

Example 1b: Establishment of 3D Culture of HepaRG Cells

[0096] HepaRG spheroids were generated in stirred-tank systems. The optimized 3D culture parameters are summarized in Table 2. Representative images of HepaRG spheroids and spheroid diameter along culture time are shown in FIG. 3. The spheroids had an average diameter of 40±7 μm, and were maintained at least for 2 weeks of culture (FIG. 3 B). The total number of HepaRG cells was maintained throughout the culture time, in contrast to what was observed for HepG2 (data not shown). The differences between 3D cultures of HepG2 and HepaRG cells can be explained by the non-proliferative phenotype of HepaRG cells in 3D, as previously reported by our team [Rebelo, S. P., Costa, R., Estrada, M., et al. (2014) HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism. Arch Toxicol.], contrary to HepG2 spheroids, which were highly proliferative in 3D culture conditions.

TABLE-US-00002 TABLE 2 Culture conditions for the establishment of 3D culture of HepaRG cells. Cell inoculum 0.3 × 10.sup.6 cell/mL concentration Culture medium William's E Medium + 2 mM L-Glutamine, 5 μg/ml Insulin + 10% (v/v) FBS + 1% (v/v) PenStrep Agitation rate 40-60 rpm Feeding regimen 20% (v/v), twice a week

Example 1c: Establishment of 3D Culture of HC-04 Cells

[0097] 3D cultures of the HC-04 cell line were established based on the conditions implemented for HepG2 cells. When HC-04 cells were cultured in 10% FBS, variation of inoculum concentration and agitation rate had no beneficial effect on cell aggregation efficiency; HC-04 cells formed very few and non-compact spheroids (FIG. 4). Increasing FBS concentration to 20% during the first three days of culture improved aggregation efficiency and generated more compact HC-04 spheroids (FIG. 4). The optimized culture strategy is summarized in Table 3. Using the optimized aggregation strategy, HC-04 cells formed compact spheroids with high cell viability (FIG. 5 A). At day 4 of culture, HC-04 spheroids presented an average diameter of 58±16 μm (FIG. 5 B), which increased throughout culturing time, reaching approximately 100±24 μm by day 9. Analysis of basal gene expression of CYP3A4 and CYP2D6 indicated that, despite the fluctuations in gene expression over the culture period, at the 2.sup.nd week of culture (day 15) there was a peak in the expression of these genes. On the other hand, CYP1A2 expression levels decrease over the culture period (FIG. 5 C).

[0098] The hepatic phenotype of HC-04 spheroids was characterized by immunofluorescence microscopy (FIG. 6). Detection of E-cadherin in the intercellular junctional spaces indicated tight cell-cell contacts, which were previously reported to be maximized in 3D cultures [Tostões, R. M., Leite, S. B., Serra, M., et al. (2012) Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing. Hepatology, 55(4), 1227-1236]. F-actin enrichment in the intercellular regions detected throughout the spheroids indicated high cellular polarization and the presence of bile canaliculi-like structures, typical of hepatic cells. Hepatic identity was further corroborated by detection of albumin, one of the liver-specific biosynthetic products and the presence of the hepatic specific protein HNF4α in all cells of the spheroid. Detection of CY3A4 (FIG. 6 E) confirmed the expression of hepatic metabolizing enzymes by HC-04 cells in spheroids, as well as of CD81, one of the receptors known to be involved in the Plasmodium entry in the hepatocytes [Foquet, L., Hermsen, C. C., Verhoye, L., et al. (2014) Anti-CD81 but not anti-SR-BI blocks Plasmodium falciparum liver infection in a humanized mouse model. Journal of Antimicrobial Chemotherapy, 70(February), 1784-1787], was detected heterogeneously within the spheroid, being mostly accumulated in the cell membranes (FIG. 6 F). Overall, HC-04 cells in spheroids present typical phenotypic hallmarks of hepatocytes.

TABLE-US-00003 TABLE 3 Culture conditions for the establishment of 3D cultures of HC-04. Cell inoculum 0.3 × 10.sup.6 cell/mL concentration Culture medium DMEM F12 + 10% (v/v) FBS + 1% (v/v) PenStrep Agitation rate 80-105 rpm Feeding regimen 50% (v/v), every other day

Example 1d: Establishment of 3D Culture of Primary Human Hepatocytes

[0099] The 3D culture of cryopreserved primary human hepatocytes (PHH) was established based on the previously described strategy for hepatic cell lines, with the same cell inoculum concentration and increasing the initial agitation speed according to Table 4. PHH spheroids were compact after 6 days of culture and the 3D culture was maintained for up to two weeks in stirred-tank vessels (FIG. 7).

TABLE-US-00004 TABLE 4 Culture conditions for the establishment of 3D culture of cryopreserved PHH. Cell inoculum 0.3 × 10.sup.6 cell/mL concentration Culture medium William's E + 10% (v/v) FBS + hepatocyte maintenance supplement* Agitation rate 80-105 rpm Feeding regimen 25% (v/v), every other day *Commercially available, recommended by PHH supplier

Example 1e: Establishment of Heterotypic Culture of Hepatic Spheroids (3D Co-Culture of HC-04:HepaRG and PHH:HepaRG)

[0100] A 3D co-culture of HC-04 and HepaRG cell lines was established based on of the aggregation conditions implemented for HC-04 cells. Cells were co-cultured in a ratio of 2 HC-04:1 HepaRG, in DMEM+F12 culture medium according to Table 3. The co-culture with HepaRG cells had a beneficial effect on cell aggregation, as compared to HC-04 mono-cultures, enabling the generation of spheroids with a FBS concentration of 10% (v/v). Variation of agitation rate from 50 to 80 rpm along two weeks of culture time led to the generation of compact spheroids (FIG. 8 A). Using the optimized aggregation strategy the resulting spheroids presented an average diameter of 65±13 at day 4 of culture, reaching approximately 113±32 μm by day 9 (data not shown).

[0101] For the co-culture of PHH with HepaRG cell line, a ratio of 9 PHH:1 HepaRG ratio at 2×10.sup.5 cell/mL cell density was tested. The aggregation was efficient, with spheroids formed 3 days after inoculation and culture viability was maintained over the culture period (FIG. 8 B). The co-culture strategy with the HepaRG cell line for both cell sources led to an effective aggregation efficiency in the first four days of culture.

II. Infection of 3D Cultures & Characterization

Example 2a: Infection of 3D Culture of HepG2 Cells with P. berghei Sporozoites in Dynamic Conditions

[0102] For the infection of a large number of spheroids and maintenance in culture for long-term periods, the infection in dynamic conditions using spinner vessels was implemented. Several parameters were considered to establish the dynamic infection, such as the sporozoite and cell concentrations, cell-to-sporozoite ratio and culture volume and agitation during infection, with the aim of maximizing cell-to-sporozoite contact and minimizing the impact of shear stress on the viability of hepatic spheroids. The parameters and conditions used for implementation of infection in dynamic conditions using spinner vessels are summarized in Table 5.

TABLE-US-00005 TABLE 5 Culture conditions for the establishment of infection in dynamic conditions. Cell line HepG2 Culture Cell concentration (cell/mL) 0.5 × 10.sup.6 parameters Volume (ml)  5 Agitation speed (rpm) 40 Infection Sp concentration (sp/mL) 0.5 × 10.sup.6 parameters Cell:sp ratio 1:1

[0103] The infection rate in dynamic conditions was assessed in 3D cultures of HepG2 and compared to static conditions using a cell-to-sporozoite ratio of 1:1, at 2.5×10.sup.4 cell/well. Cell viability 48 h post-infection was high, indicating that the manipulation of culture parameters and resulting shear stress had no impact on spheroid integrity and viability (FIG. 9 A). Moreover, the infection in dynamic conditions was successful (66% comparing with infection in static conditions), indicating that this strategy can be applied for the infection of spheroids. Since the infection in dynamic conditions requires a large quantity of sporozoites, the subsequent examples entailing infection of other hepatic cell sources and characterization of infection were performed in static conditions.

Example 2b: Infection of 3D Culture of HepG2 Cells with P. berghei Sporozoites in Static Conditions

[0104] Infection parameters including cell concentration, cell-to-sporozoite ratio, cell-to-sporozoite mode of contact and culture time of the spheroids were optimized. Sporozoites were obtained from the dissection of the salivary glands of infected Anopheles stephensi mosquitoes. Following mechanical disruption of salivary glands, the sporozoite suspension was kept on ice for up to 3 hours, until sporozoites were employed to inoculate the cells in culture.

[0105] For the implementation of standard infection conditions in spheroids, the effect of centrifugation and subsequent static culture in 96-well plates was assessed. All centrifugation speeds led to spheroid fusion except in the condition where the centrifugation speed was gradually reached and gradually decreased (equivalent to acceleration and braking profiles 5 in a Rotina420R, Hettich centrifuge), FIG. 10. Under this condition, spheroids retained integrity, without fusion, and maintained high cell viability independently of cell concentration, in the central rows of the plate (rows C, D and E). Thus, culture progression was evaluated for 48 hours after centrifugation using the setting described. Readouts were cell viability and spheroid fusion (FIG. 11). After 48 hours in culture, HepG2 spheroids maintained high cell viability with minimal spheroid fusion in the three cell concentrations tested.

[0106] Initially, a preliminary assay employing reporter lines of Plasmodium berghei, constitutively expressing luciferase (Pb-Luc) or GFP (Pb-GFP), was performed to optimize the range of cell-to-sporozoite ratios and mode of contact. Pb-Luc parasites enable measuring infection by luminescence readings of cell lysates following addition of the luciferin substrate. Pb-GFP parasites enable measuring infection flow cytometry analysis. Such analyses allow measuring the percentage of invaded cells (% GFP-positive cells) and the development of the parasite inside the hepatic cells (GFP intensity). The conditions tested and readouts employed are depicted in Table 6 and the results obtained are presented in FIG. 12.

TABLE-US-00006 TABLE 6 Parameters tested for optimization of infection of HepG2 spheroids with Pb-Luc and the correspondent readout. Conditions Tested Readout 2D vs 3D Infection 2.sup.nd week spheroids Cell densities - 0.5, 1, 1.5, 2 and rate:Luciferase 2.5 × 10.sup.4 cell/well activity Cell:sporozoite ratio - 1:2, 1:1, 3:2, 2:1 and 5:2 (luminescence- Centrifuged (1800 xg) vs. non-centrifuged based assay)

[0107] HepG2 spheroids presented higher infection rate when cell-to-sporozoite contact was promoted by centrifugation (FIG. 12 A). In these conditions, the highest infection rates were obtained for cell-to-sporozoite ratios of 1:2 and 1:1, using the cell concentrations of 2.5 and 5×10.sup.4 cell/well (FIG. 12 B).

[0108] Therefore, the preferred procedure for infection was: (i) Distribution of spheroids from spinner vessel to 96 well plates for infection; (ii) Promotion of sporozoite-to-cell contact by centrifugation at 1800×g for 5 min with medium acceleration and braking; (iii) Maintenance of spheroids in 96-well plates, in static conditions, for 48 hours post-infection, for infection assessment.

[0109] Cell-to-sporozoite ratios of 1:2 and 1:1 were selected to proceed with the optimization of P. berghei infection. Aiming to maximize cell-to-sporozoite contact, cell density at infection was optimized to achieve the maximum coverage of the well surface. The results are presented in FIG. 13. Inoculation of 2.5×10.sup.4 and 5×10.sup.4 cell/well (7.8×10.sup.4 and 15.6×10.sup.4 cell/cm.sup.2, respectively) led to 60-80% of the well surface covered by spheroids, with no spheroid fusion being observed after 48 hours in culture. Conversely, when using a seeding density of 10×10.sup.4 cell/well, spheroid fusion occurred. Thus, the lower cell concentrations (2.5×10.sup.4 and 5×10.sup.4 cell/well) were selected for further optimization of HepG2 spheroids infection with P. berghei, by evaluating two cell-to-sporozoite ratios (1:1 and 1:2) and using spheroids generated by 1 or 2 weeks in culture.

[0110] The results showed that higher infection rates were obtained with hepatic spheroids generated by two weeks in culture (Table 6). Moreover, the infection rates could be maximized using 5×10.sup.4 cell/well and a 1:2 cell-to-sporozoite ratio for both lines of P. berghei (150% and 80% relative to HepG2 2D cells, for Pb-Luc and Pb-GFP respectively; FIG. 14 A; B). Nonetheless, 2.5×10.sup.4 cell/well in 1:2 cell-to-sporozoite ratio may be considered as alternative in case of limited sporozoite availability, leading to a Pb-Luc infection rate of 89 relative to 2D cultures (FIG. 14 A; B). In general, the infection rate of HepG2 spheroids is comparable to the obtained for 2D cells (FIG. 14 A; B).

TABLE-US-00007 TABLE 6 Spheroids in 1.sup.st week of culture vs 2.sup.nd week of culture. Infection rate represented as luciferase activity normalized to that of HepG2 2D cultures infected in 1:1 cell-to-sporozoite ratio. Results from at least two independent experiments, except for 5 × 10.sup.4 cell/well in 1:2 cell-to-sporozoite ratio. Cell:Pb Cell density Infection rate (% to 2D) ratio (10.sup.4 cell/well) Spheroids 1.sup.st week Spheroids 2.sup.nd week 1:1 2.5 15.2 ± 10.9 21.9 ± 8.5 1:2 26.1 ± 13.8  78.9 ± 23.5 1:1 5 46.4 ± 0.9   63.6 ± 51.6 1:2 43.6 140.5 ± 82.7

[0111] Both analytical methods, assessment of luciferase activity or GFP fluorescence, were consistent in identifying the infection conditions leading to the highest infection rates (FIG. 14 A; B). The differences in the infection rates observed might be explained by the differences inherent to the two analytical methods employed. For Pb-GFP, infection rate reflects the number of infected cells (percentage of GFP-positive cells) or the development of the parasite (GFP intensity). Conversely, infection with Pb-Luc is analyzed by luciferase activity, and infection rates cumulatively reflect the number of infected cells as well as the development of the Pb-Luc parasite.

[0112] Sporozoites were able to develop in HepG2 spheroids, presenting, in all the conditions employed, a development above 65% of that observed in 2D cultures (FIG. 14 C) and higher for the lower cell density conditions (2.5×10.sup.4 cell/well).

[0113] Given the data obtained, an optimal strategy for P. berghei infection of HepG2 spheroids was implemented using: (i) spheroids from two-week cultures; (ii) a cell density of 5×10.sup.4 cell/well; and (iii) a 1:2 cell-to-sporozoite ratio.

Example 2c: Infection of 3D Culture of HC-04 Cells with P. berghei Sporozoites in Static Conditions

[0114] HC-04 cells were infected by both P. berghei parasite lines. In 2D cultures, the infection rate of HC-04 cells was approximately 79% and 47% of the one observed for HepG2 cells under 2D conditions for Pb-Luc and Pb-GFP, respectively (FIG. 15 A; B). Like for HepG2 cells, P. berghei infection was optimized in HC-04 spheroids by evaluating different (i) cell-to-sporozoite ratios and (ii) cell densities. The infection rate could be maximized using a cell-to-sporozoite ratio of 1:2 and a cell density of 5×10.sup.4 cell/well (FIG. 15 A; B), similarly to what was described above for HepG2 (FIG. 14 A; B). For all conditions tested, Pb-GFP development in 3D cultures of HC-04 was comparable or higher than in 2D cultures of HepG2 (FIG. 15 C).

[0115] The percentage of spheroids infected by Pb-GFP at 1:2 of cell-to-sporozoite ratio was quantified by fluorescence microscopy, for cell densities of 2.5×10.sup.4 and 5×10.sup.4 cell/well. In both conditions more than 55% of the spheroids were infected (FIG. 16 A), with an average of approximately 3 infected cells per infected spheroid (FIG. 16 B).

Example 2d: Assessment of Parasite Development Over Time in 3D Cultures

[0116] In addition to the implementation and optimization of P. berghei infection in 3D, the characterization of parasite development was performed for both hepatic cell lines (HepG2 and HC-04). Parasite development observed 60 hours post-infection was characterized by quantification of GFP intensity. A comparable profile of development was observed for all the conditions tested (2D and 3D; HepG2 and HC-04) (FIG. 17 A). P. berghei development dynamics over showed an increasing profile reaching its maximum value at 48 h, after which is maintained up to 60 h post-infection (FIG. 17 A). Concomitantly, Pb-GFP was able to replicate inside hepatic cells that have been effectively invaded, leading to an increase in the size of the infected hepatic cells both in 2D and 3D (FIG. 17 B). Moreover, the detection of UIS4 at 48 hours post-infection, a protein in the parasitophorous vacuole membrane, confirmed the parasite development inside the parasitophorous vacuole (FIG. 17 C). To assess whether the parasite development in 3D cultures is complete, the release of merosomes was evaluated and in both 2D and 3D cultures from both cell lines (HepG2 and HC-04) there was detection of merosomes in the culture supernatant at 72 h post-infection. The culture supernatant containing merosomes was injected into mice and parasitemia was monitored over time by evaluating the percentage of infected red blood cells (RBC) (FIG. 18). Parasitemia was detected in mice for all the conditions tested, showing that the sporozoite development in 2D and 3D cultures is comparable and results in mature merosomes containing infective merozoites.

Example 2e: Infection of 3D Co-Culture of HC-04 Cells and HepaRG with P. berghei Sporozoites

[0117] The characterization of HC-04 metabolic activity and its suitability to be used as an in vitro model for drug screening is scarce. This may represent a major limitation for anti-Plasmodium drug assessment in this model, given the importance of liver metabolic activity for the correct metabolization of some anti-Plasmodium drugs (e.g, primaquine). In order to overcome this limitation, strategies based on co-culture systems were considered. Here, HepaRG cell line was selected to pursue a co-culture strategy, since these cells have been previously described as a more accurate surrogate of liver function among the available human hepatic cell lines platforms [Rebelo, S. P., Costa, R., Estrada, M., et al. (2014) HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism. Arch Toxicol.]. Moreover, previous reports have shown that co-cultures of primary hepatocytes and HepaRG could extend hepatocyte integrity and fitness, as well as improve P. cynomolgi infection [Dembélé, L., Franetich, J., Lorthiois, A., et al. (2014) Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures. Nature Medicine, 20(3), 307-312].

[0118] It was assessed whether the co-culture of HC-04 and HepaRG would have an impact on P. berghei infection. A 3:1 ratio of HC-04 to HepaRG cells was tested. Infection of co-cultures and HC-04 monocultures were performed with Pb-GFP in the optimized conditions described above (two weeks spheroids, cell density 2.5×10.sup.4 and 5×10.sup.4 cell/well in a 1:2 ratio). The results are presented in Table 7.

TABLE-US-00008 TABLE 7 Plasmodium infection of HC-04:HepaRG spheroids. Infection by Pb-GFP represented as the frequency of GFP-positive cells. Data from a single experiment. Cell:Pb Cell density Infection rate (% of GFP+ cells) ratio (10.sup.4 cell/well) Monoculture Co-culture 1:2 2.5 0.56 0.19 5 0.37 0.40

[0119] The results indicate that co-culture did not influence the infection rate in the best condition identified for infection of HC-04 spheroids (1:2 cell-to-sporozoite ratio and cell density of 5×10.sup.4 cell/well). Thus, this co-culture strategy constitutes a promising alternative to improve the metabolic capacity of the system, as compared to HC-04 monocultures.

III. In Vitro Testing of Reference Drugs Against Infected of 3D Cultures

Example 3: Test of Reference Anti-Plasmodium Drugs Primaquine and Atovaquone

[0120] The suitability of the platform presented in this invention for drug screening purposes of anti-infective agents was explored using one reference drug, Atovaquone (ATQ), requiring no metabolization to target the liver-stage Plasmodium infection.

[0121] HC-04 3D cultures were infected with Pb-Luc in the optimized conditions described above (cell density of 2.5×10.sup.4 cell/well in a 1:2 ratio). The assessment of drug effect in the infection was performed by incubating the drug at a range of concentrations from 0.01 to 100 nM for 1 hour before incubation with the sporozoites and the readout was performed 48 hours after sporozoites addition, described as incubation regimen (A) in the detailed description of the invention section (FIG. 1). The drug concentrations employed were shown not to affect cell viability. A dose response curve was established for the 3D cultures treated with ATQ and a 0.6 nM half inhibitory concentration (IC.sub.50) for sporozoite infection was determined. The highest concentrations tested led to a decrease of more than 90% of infection (FIG. 19). ATQ performed similarly in 2D and in 3D cultures (data not shown).