Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration

Summary

R-spondin1 plays a pivotal role in potentiating canonical WNT/β-catenin signaling, a pathway critical for maintaining stem cell homeostasis and driving tissue regeneration. Here, we present a scalable, serum-free protocol for producing R-spondin1-loaded small extracellular vesicles (evRSPO1) from engineered HEK293 cells, with multimodal characterization of vesicle quality and bioactivity. We also describe steps for in vitro functional validation of the vesicles using mouse intestinal organoids and for in vivo therapeutic applications in mouse intestinal regeneration via oral delivery.

For complete details on the use and execution of this protocol, please refer to Yang et al.^1,2

Graphical abstract

Highlights

• •Scalable production of R-spondin1-loaded small extracellular vesicles (evRSPO1)
• •Steps for in vitro multimodal characterization of purified evRSPO1
• •Instructions for functional validation of WNT pathway activation by evRSPO1
• •Guidance for oral delivery of evRSPO1 in mouse intestinal injury and aging models

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

R-spondin1 plays a pivotal role in potentiating canonical WNT/β-catenin signaling, a pathway critical for maintaining stem cell homeostasis and driving tissue regeneration. Here, we present a scalable, serum-free protocol for producing R-spondin1-loaded small extracellular vesicles (evRSPO1) from engineered HEK293 cells, with multimodal characterization of vesicle quality and bioactivity. We also describe steps for in vitro functional validation of the vesicles using mouse intestinal organoids and for in vivo therapeutic applications in mouse intestinal regeneration via oral delivery.

Before you begin

The protocol below describes the specific steps for using male C57BL/6J mice (6–8 weeks old for injury models, 18 months old for aging studies) and male Axin2-mGFP mice (6–8 weeks old). We have also used this protocol with C57BL/6J mice of both sexes and other intestinal injury models, including DSS-induced colitis. Specific evRSPO1 qualitative analysis experiments, including NanoFCM particle concentration, ELISA loading quantification, and TOPFlash reporter assay, were performed at least three times, in triplicate. The variation between batches being less than 15% is considered acceptable. All steps are optimized for laboratory-scale production of evRSPO1 and preclinical testing in mice; scale-up for industrial production requires additional process validation.

Innovation

RSPO1 is recognized as a major driver of WNT-dependent crypt self-renewal with therapeutic potential through promoting gastrointestinal tissue regeneration.³ This protocol establishes a robust, scalable workflow for oral delivery of functional RSPO1 via small extracellular vesicles (sEV),^4,5 overcoming longstanding translational barriers of free RSPO1 protein, including gastric degradation, low stability, and poor intestinal targeting. Key innovations include: (1) a serum-free suspension culture system for high-yield production of bioactive evRSPO1, with > 80% activity retention after 8 weeks of storage; (2) a non-genetic loading strategy that anchors RSPO1 to the sEV surface via heparan sulfate proteoglycans, preserving full WNT-potentiating activity; (3) a validated oral delivery protocol that enables targeted RSPO1 delivery to mouse intestinal crypts, with no off-target histopathological changes in major organs; and (4) a complete functional validation pipeline from in vitro organoid assays to in vivo preclinical efficacy testing in injury and aging models. This protocol outperforms existing recombinant RSPO1 delivery methods by enabling oral administration, a critical advancement for clinical translation of intestinal regenerative therapies.

Institutional permissions

All animal experimental procedures were approved by the Ethics Committee of Guangzhou National Laboratory (AUCP-2022-12-A02) and performed under the Guide for the Care and Use of Laboratory Animals.

Preparation of pEF1A-hRSPO1-His-PuroR plasmid

Timing: 1 week

• 1.
  Prepare pEF1A-hRSPO1-His-PuroR plasmid.

  • a.
    Human RSPO1 fused to a C-terminal 6×His tag was synthesized by Igebio and subcloned into the pLVX-MSC-IRES-PuroR plasmid via the EcoRI and XbaI restriction sites under the control of the EF1α promoter (Figure 1).
  • b.
    Use the primer EF1A-F (5′-CTGGGAAAGTGATGTCGTGT-3′) for Sanger sequencing to confirm the integrity of the inserted sequence.

Figure 1.

Vector construction for RSPO1-overexpression

(A) Protein sequence for human RSPO1 with 6×His tag.

(B) Vector map for pEF1A-hRSPO1-His-PuroR.

Preparation of RSPO1 protein overexpression lentiviruses

Timing: 1 week

• 2.
  Prepare RSPO1-overexpression lentiviruses.

  • a.
    Culture HEK293T cells in high-glucose DMEM medium with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS) at 37°C and 5% CO₂.
  • b.
    Seed HEK293T cells in a 10-cm culture dish to reach 70%–80% confluency the next day.
  • c.
    Replace with fresh media (10 mL for a 10-cm dish) and culture for 20 min before transfection.
  • d.
    Add 9.58 μg pEF1A-hRSPO1-His-PuroR plasmid and co-transfection plasmid (4.66 μg psPAX2 and 3.99 μg VSVG) and 24 μL Lipo8000 in 750 μL Opti-MEM Medium.
  • e.
    Mix well, and incubate for 20 min in the dark at room temperature (RT).
  • f.
    Add the mixture dropwise to the cells, and culture in the incubator.
  • g.
    Collect lentivirus supernatants at 24 h and 48 h post-transfection (the total volume is approximately 20 mL).

    • i.
      Centrifuge at 4°C for 5 min.
    • ii.
      Filter with a 0.45-μm filter to remove cell debris.

Note: The lentivirus supernatants can be stored at −80°C for up to 6 months.

Preparation RSPO1 protein-overexpression HEK293 cells

Timing: Approximately 15 days

• 3.
  Lentivirus infection.

  • a.
    Culture HEK293 cells in high-glucose DMEM medium with 10% FBS and 1% PS at 37°C and 5% CO₂.
  • b.
    Seed HEK293 cells in a 35-mm culture dish to reach 30%–50% confluency the next day.
  • c.
    Add 1 mL lentivirus supernatant to the cells in the presence of 2 μg/mL polybrene.

    • i.
      Incubate for 24 h.
    • ii.
      Replace the medium with fresh culture medium.
  • d.
    Repeat step 3.c another time.
• 4.
  Stable cell selection.

  • a.
    Replace the media with fresh culture medium containing 2 μg/mL puromycin for stable cell selection.
  • b.
    Refresh the puromycin-containing media every 3 days for 7–10 days, until all non-infected control cells are removed.
  • c.
    Dilute the puromycin-resistant cells to a density of 1 cell/100 μL in fresh culture medium containing 2 μg/mL puromycin.
  • d.
    Seed the diluted cell suspension into 4 × 96-well plates at 100 μL/well.
  • e.
    Incubate the plates at 37°C with 5% CO₂ for 10–14 days until single monoclonal colonies form.
  • f.
    Collect the culture supernatant from each monoclonal cell line.
  • g.
    Measure WNT/β-catenin signaling activity of the supernatant via TOPFlash luciferase reporter assay (related to step 9).
  • h.
    Select the monoclonal cell line with the highest TOPFlash activity.
  • i.
    Expand the selected cell line in high-glucose DMEM medium with 10% FBS and 1% PS at 37°C and 5% CO₂.
  • j.
    Validate RSPO1 expression in the selected cell line via RT-qPCR and Western blot.

Note: The ΔΔCt of RSPO1 gene expression should be ≥ 10.

Preparation of TOPFlash luciferase reporter assay cells and luciferase assay

Timing: 5 days

• 5.
  Prepare Topflash-pEF1A-IRES-PuroR plasmids.

  • a.
    DNA Fragments containing 7×TCF/LEF binding element sites were amplified from M50 Super 8×TOPFlash plasmid, and subcloned into pLVX-MSC-IRES-PuroR plasmid using seamless assembly (Figure 2).
  • b.
    The integrity of the inserted sequence was confirmed via Sanger sequencing using primers: 5′-CCCCTGAACCTGAAACATAAAAT-3′, 5′-GTAGATCCAGAGGAGTTCATG-3′, and 5′-GCGGTTCCATCTTCCAG-3'.
• 6.
  Transfection.

  • a.
    Culture HEK293T cells in high-glucose DMEM medium with 10% FBS and 1% PS at 37°C and 5% CO₂.
  • b.
    Seed HEK293T cells in a 35-mm culture dish for 18–24 h until cells reach 30%–50% confluency.
  • c.
    Replace the medium with fresh culture medium and incubate for 20 min before transfection.
  • d.
    Add 9.58 μg Topflash-pEF1A-IRES-PuroR plasmid and co-transfection plasmid (4.66 μg psPAX2 and 3.99 μg VSVG) and 24 μL Lipo8000 in 750 μL Opti-MEM Medium
  • e.
    Mix well, and incubate for 20 min in the dark at RT.
  • f.
    Add the mixture dropwise to the cells, and culture in the incubator.
  • g.
    Collect lentiviral supernatants at 24 h and 48 h post-transfection (the total volume is approximately 20 mL).
  • h.
    Centrifuge at 4°C for 5 min.
  • i.
    Filter with a 0.45-μm filter to remove cell debris

Note: The lentivirus supernatants can be stored at −80°C for up to 6 months.

• 7.
  Infection and stable cell selection.

  • a.
    Culture HEK293 cells in high-glucose DMEM medium with 10% FBS and 1% PS at 37°C and 5% CO₂.
  • b.
    Seed HEK293 cells in a 35-mm culture dish for 18–24 h until cells reach 30%–50% confluency.
  • c.
    Add 1 mL lentivirus supernatant to the cells in the presence of 2 μg/mL polybrene.
  • d.
    Incubate for 24 h. Replace the medium with fresh culture medium.
  • e.
    Repeat step 7.c another time.
  • f.
    Replace the medium with culture medium containing 2 μg/mL puromycin for stable cell selection.
  • g.
    Refresh the puromycin-containing medium every 3 days for 7–10 days, until all non-infected control cells are eliminated.
• 8.
  Cell treatment.

  • a.
    Seed the selected cells in a 96-well plate at a density of 30,000 cells per well and culture overnight.
  • b.
    Refresh the media with high-glucose DMEM containing 10% FBS and 1% PS, supplemented with EVs or recombinant protein, at 37°C and 5% CO₂ for 24 h.
• 9.
  Luciferase detection.

  • a.
    Prepare 1×passive lysis buffer (PLB) by diluting 1 volume of 5×PLB with 4 volumes of ddH₂O.

    Note: Ensure the buffer is at RT before use.

  • b.
    Aspirate the growth medium from the 96-well plate and wash cells once with PBS.
  • c.
    Add 50 μL of 1×PLB to each well.
  • d.
    Shake the plate at 1,000 rpm on an orbital shaker for 30 min at RT to ensure complete cell lysis.
  • e.
    Add 70 μL of Firefly Luciferase Assay Reagent (containing luciferin substrate) to each well.
  • f.
    Immediately measure the luminescence (RLU) using a microplate reader.

    Note: Integration Time: Set to 1–10 s per well (typically 2 s). Delay Time: Set a 2 s delay after injection/addition before measurement if using an auto-injector.

Figure 2.

Vector construction for Topflash-pEF1A-IRES-PuroR

(A) Nucleotide sequence for 7×TCF/LEF binding elements driving expression of firefly luciferase.

(B) Protein sequence for firefly luciferase.

(C) Vector map for Topflash-pEF1A-IRES-PuroR.

Preparation of mouse intestinal organoid

Timing: 1 week

• 10.Prepare the medium for intestinal organoid culture by adding all supplements to the base medium following the final concentrations indicated in the “Materials” table below.

Note: In the intestinal organoid medium, 500 ng/mL human recombinant RSPO1 protein is added as the positive control. Withdraw the RSPO1 protein and add evRSPO1 to a final concentration of 1 × 10⁹ particles/mL.

• 11.
  Prepare intestinal crypts.

  • a.
    Euthanize 6-8-weeks-old male C57BL/6J mice via cervical dislocation.
  • b.
    Excise the entire small intestine from the gastroduodenal junction to the ileocecal valve.
  • c.
    Use a 50 mL syringe along with a gastric lavage needle to draw 10 mL of ice-cold sterile PBS to flush the intestinal lumen and remove fecal contents.

    Note: This process should be repeated three to four times.

  • d.
    Longitudinally open the intestine and cut into 5–10 mm fragments.
  • e.
    Wash the intestinal fragments 5 times with ice-cold PBS.
  • f.
    Incubate the fragments in 2 mM EDTA/PBS solution at 4°C for 30 min with gentle rotation at 20–30 rpm.

    CRITICAL: Strictly control the EDTA incubation time to avoid crypt damage and reduced viability.

  • g.
    Remove the EDTA solution and add 10 mL of ice-cold PBS.
  • h.
    Vortex the fragments vigorously for 10 s to release crypts.
  • i.
    Filter the crypt-enriched suspension through a 70-μm cell strainer.
  • j.
    Centrifuge the filtrate at 300 g for 3 min at 4°C.
  • k.
    Resuspend the crypt pellet in 0.1% BSA/PBS for counting.
• 12.
  Establish intestinal organoid cultures.

  • a.
    Resuspend isolated crypts in ice-cold growth factor-reduced Matrigel at a density of 200–500 crypts/20 μL.
  • b.
    Plate 20 μL Matrigel domes in the center of each well of a 24-well plate.
  • c.
    Polymerize the Matrigel at 37°C for 10 min.
  • d.
    Add 500 μL pre-warmed intestinal organoid medium to each well.
  • e.
    Incubate the plates at 37°C with 5% CO₂.
  • f.
    Refresh the medium every 3 days.
  • g.
    Pass the organoids every 7 days via mechanical dissociation in cold PBS, centrifugation, and re-embedding in fresh Matrigel.
• 13.
  Treat intestinal organoids with evRSPO1.

  • a.
    Dissociate organoids into single cells via mechanical pipetting and incubation with TrypLE Select for 5 min at 37°C.
  • b.
    Centrifuge the single-cell suspension at 300 g for 3 min at 4°C.
  • c.
    Resuspend the cells in Matrigel and plate in 24-well plates.
  • d.
    After Matrigel polymerization, add 500 μL intestinal organoid medium without hrRSPO1 to each well.
  • e.
    Add treatment groups to the medium: 1×10⁹ particles/mL evRSPO1, 1×10⁹ particles/mL evWT, 500 ng/mL hrRSPO1, or medium without RSPO1 (negative control).
  • f.
    Incubate the plates at 37°C with 5% CO₂ for 48 h.
• 14.
  Assess evRSPO1 cellular uptake by intestinal organoids.

  • a.
    Label evRSPO1 with PKH26 fluorescent dye.
  • b.
    Repurify the labeled evRSPO1 via the EXODUS system to remove residual unbound dye.
  • c.
    Add PKH26-labeled evRSPO1 to organoid cultures for 12 h.
  • d.
    Fix the organoids with 4% paraformaldehyde (PFA) for 30 min at RT.
  • e.
    Permeabilize the organoids with 0.3% Triton X-100 for 10 min.
  • f.
    Stain the organoids with FITC-Phalloidin and DAPI for 30 min at RT.
  • g.
    Wash the organoids 3 times with PBS.
  • h.
    Image the organoids via confocal laser scanning microscopy.
• 15.
  Evaluate WNT signaling activation in organoids via fluorescence imaging of Axin2-mGFP signals.

  • a.
    Capture fluorescence images of Axin2-mGFP in treated organoids via confocal microscopy.
  • b.
    Quantify Axin2-mGFP fluorescence intensity via ImageJ.
• 16.
  Quantify organoid growth and crypt budding.

  • a.
    Capture brightfield images of treated organoids.
  • b.
    Count the number of crypt-villus budding structures per organoid.
  • c.
    Analyze 3 independent fields of view per biological replicate.

Note: Include only organoids with a diameter > 200 μm.

• 17.Perform molecular validation of WNT pathway activation and stem cell proliferation via RT-qPCR and Western blot.

Note: Molecular validation including WNT target genes (Lgr5, Axin2, Myc, Ccnd1), proliferation markers (Ki67, Pcna), and cell type-specific markers (Olfm4, Ascl2, Muc2, Lyz1).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Ms anti-β-actin (1:5000)	Cell signal	Cat. 3700S
Ms anti-R-Spondin1 (1:2000)	R&D systems	Cat. MAB4658
Ms anti-His-tag (1:1000)	Cell signal	Cat. 2366
Rb anti-Non-phospho (Active) β-Catenin (Ser45) (1:1000)	Cell signal	Cat. 19807S
Rb anti-β-Catenin (1:1000)	Cell signal	Cat. 8480S
Rb anti-TSG101 (1:2000)	Abcam	Cat. ab125011
Rb Anti-CD63 (1:2000)	Abcam	Cat. ab134045
Rb Anti-CD9 (1:1000)	Abcam	Cat. ab236630
Rb anti-Calnexin (1:1000)	Abcam	Cat. ab133615
Rb anti-HSP70 (1:1000)	Abcam	Cat. ab181606
Rb anti-Ki67 (1:1000)	Abcam	Cat. ab15580
Rb anti-Alix (1:200)	Abcam	Cat. ab275377
Rb anti-p21 (1:200)	Abcam	Cat. ab188224
Rb anti-Olfm4 (1:500)	Cell signal	Cat. 39141S
Rb anti-CD45 (1:500)	Abcam	Cat. ab10558
Ms anti-IFNγ (1:500)	Santa Cruz	Cat. Sc-8423
Ms anti-ZO-1 (1:500)	Invitrogen	Cat. XL361204
Rb anti-Occludin (1:500)	Abcam	Cat. ab216327
Rb anti-Muc2 (1:500)	Abcam	Cat. ab272692
Alexa Fluor 647-conjugated anti-CD63 antibody (1:200)	Abcam	Cat. ab233056
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (1:200)	Invitrogen	Cat. A-11008
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (1:200)	Invitrogen	Cat. A-11012
HRP-conjugated Goat anti-Rabbit antibody (1:1000)	Beyotime	Cat. A0208
HRP-conjugated Goat anti-Mouse antibody (1:1000)	Beyotime	Cat. A0216
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium (DMEM)	Cytiva	Cat. SH20243.01
Fetal bovine serum	Corning	Cat. 35-179-CV
Penicillin/streptomycin	Gibco	Cat. 15140-122
Puromycin	Gibco	Cat. A1113803
TrypLE Select	Gibco	Cat. A1217701
Serum-free medium for HEK293	Bioengine	Cat. TP0103001
Opti-MEM	Gibco	Cat. 11058021
psPAX2	Addgene	Cat. 12260
VSVG	Addgene	Cat. 8454
M50 Super 8×TOPFlash	Addgene	Cat. 12456
Lipo8000	Beyotime	Cat. C0533
Polybrene	MedChem	Cat. HY-K2032
Goat Anti-Mouse Ig (10nm Gold)	Abcam	Cat. ab39619
Heparinase I and III Blend	Sigma	Cat. H3917
Growth factor-reduced Matrigel	Corning	Cat. 356231
Advanced DMEM/F12	Gibco	Cat. 12634028
EGF recombinant protein	Novoprotein	Cat. C029
Noggin recombinant protein	Novoprotein	Cat. CB89
RSPO1 recombinant protein	Novoprotein	Cat. CX83
N2	Gibco	Cat. 17502048
B27	Gibco	Cat. 17504044
GlutaMAX	Gibco	Cat. 35050061
N-acetylcysteine	Sigma	Cat. A0737
PKH26	Sigma	Cat. MKCV7372
DAPI	Sigma	Cat. D9542
FITC-Phalloidin	Beyotime	Cat. C2221S
Trizol	Invitrogen	Cat. 15596018CN
DiR	Meilun Bio	Cat. MB12482
RIPA lysis buffer	Beyotime	Cat. P0013B
Protease inhibitors	Beyotime	Cat. P1005
5×loading buffer	Beyotime	Cat. P0015L
Uranyl acetate	MedChem	Cat. HY-184835
Glutaraldehyde	MedChem	Cat. HY-Y1017
BSA	Sangon	Cat. A602448
20×TBS buffer	Sangon	Cat. B548105
Tween-20	Sangon	Cat. A600560
0.22-μm Filter Unit	Millipore	Cat. SLGPR33RB
0.45-μm Filter Unit	Millipore	Cat. SLHVR33RS
70-μm cell strainer	BD Biosciences	Cat. 352350
0.45-μm PVDF membrane	Millipore	Cat. IPVH00010
96-well plate	Costar	Cat. 3799
24-well plate	Falcon	Cat. 353047
6-well plate	Falcon	Cat. 353046
10-cm dish	Corning	Cat. 430167
35-mm dish	Corning	Cat. 430165
250-mL Erlenmeyer Flasks	Biofl	Cat. TAB102250
Critical commercial assays
Dual-Luciferase Report Assay System	Promega	Cat. E1501
RSPO1 enzyme-linked immunosorbent assay kit	R&D Systems	Cat. DY4645-05
High Sensitivity ECL Substrate Kit	Abcam	Cat. ab133406
β-Galactosidase staining	Servicebio	Cat. G1073
BCA assay kit	Beyotime	Cat. P0010
HiScript II cDNA kit	Vazyme	Cat. R233-01
SYBR qPCR Master Mix	Vazyme	Cat. Q711-03
mRNA Library Fast kit	Epibiotek	Cat. R1810
Experimental models: Cell lines
HEK293 cells	Procell system	CL-0001
HEK293T cells	Procell system	CL-0005
Experimental models: Organisms/strains
C57BL/6J mice (6–8 weeks)	Gempharmatech	N/A
C57BL/6J mice (18 months)	Gempharmatech	N/A
Axin2-mGFP mice (6–8 weeks)	Yi Arial Zeng Lab	N/A
Other
Automatic cell counter	NanoEntek, Korea	N/A
EXODUS	Huixin, China	N/A
TEM	Thermo, USA	N/A
Flow Nano Analyzer	Nanofcm, China	N/A
RS2000 biological irradiator	Rad Source, China	N/A
Slide viewer	Olympus, Japan	N/A
Abbelight SAFe 180	Abbelight, France	N/A
IVIS Lumina imager	PerkinElmer, USA	N/A
Cryostat	Leica, Germany	N/A

Materials and equipment

Intestinal organoid medium

Reagent	Final concentration
Advanced DMEM/F12	N/A
RSPO1 recombinant protein	500 ng/mL
EGF recombinant protein	50 ng/mL
Noggin recombinant protein	100 ng/mL
N2	1×
B27	1×
GlutaMAX	1×
N-acetylcysteine	1 mM
penicillin/streptomycin	1%

CRITICAL: The complete medium should be stored at 4°C for up to 2 weeks (or aliquoted at −20°C for longer storage, with a maximum of 3 months).

HEK293 and HEK293T culture media

Reagent	Final concentration
DMEM	N/A
FBS	10%
penicillin/streptomycin	1%

CRITICAL: The complete medium should be stored at 4°C for up to 4 weeks.

HEK293 and HEK293T serum-free culture media

Reagent	Final concentration
Serum-free medium	N/A
penicillin/streptomycin	1%

CRITICAL: The complete medium should be stored at 4°C for up to 4 weeks.

TBST

Reagent	Final concentration
Tris	10 mM
NaCl	150 mM
Tween-20	0.1%

CRITICAL: The TBST should be stored at RT for up to 4 weeks after adding the Tween-20.

Step-by-step method details

Initiating and maintaining RSPO1-engineered cells in a chemically defined suspension medium

Timing: 3–5 weeks

In this step, we describe a step-wise transition of the anchorage-dependent FBS-cultured RSPO1-engineered HEK293 cells to a serum-free, chemically defined suspension culture. The culture medium containing animal-derived components (such as FBS and KSR) significantly affects sEV purity.

• 1.
  Initiate the suspension-cultured cells with FBS-free medium.

  • a.
    Seed the RSPO1-engineered HEK293 cells in a 10 cm dish with 10% FBS and 1% PS.
  • b.
    When the cell confluency reaches 90%, collect the cells with 0.25% Trypsin.
  • c.
    Seed the cells at 2–5×10⁵ cells/mL in 30 mL serum-free, chemically defined suspension HEK293 cell culture medium with 10% FBS in a 100 mL Erlenmeyer flask.
  • d.
    Culture on a shaker at 100 rpm for 2–3 days.
  • e.
    When the cell density reaches 1–2×10⁶ cells/mL.

    • i.
      Centrifuge at 170 g for 3 min.
    • ii.
      Remove the supernatant completely.
    • iii.
      Seed the cells at 2–5×10⁵ cells/mL in fresh suspension culture medium containing 5% FBS.
    • iv.
      Culture for 2–3 days.
  • f.
    When the cell density reaches 1–2×10⁶ cells/mL

    • i.
      Centrifuge at 170 g for 3 min.
    • ii.
      Remove the supernatant completely.
    • iii.
      Seed the cells at 2–5×10⁵ cells/mL in fresh suspension culture medium containing 2.5% FBS.
    • iv.
      Culture for 2–3 days.
  • g.
    When the cell density reaches 1–2×10⁶ cells/mL.

    • i.
      Centrifuge at 170 g for 3 min.
    • ii.
      Remove the supernatant completely.
    • iii.
      Seed the cells at 2–5×10⁵ cells/mL in fresh suspension culture medium containing 1% FBS.
    • iv.
      Culture for 2–3 days.
  • h.
    When the cell density reaches 1–2×10⁶ cells/mL.

    • i.
      Centrifuge at 170 g for 3 min.
    • ii.
      Remove the supernatant completely.
    • iii.
      Seed the cells at 2–5×10⁵ cells/mL in fresh suspension culture medium.
    • iv.
      Culture for 2–3 days.
• 2.
  Maintain the suspension-cultured cells at high cell density.

  • a.
    When the cell density reaches 1-2x10⁶ cells/mL.

    • i.
      Centrifuge at 170 g for 3 min.
    • ii.
      Remove the supernatant completely.
    • iii.
      Seed the cells at 2–5×10⁵ cells/mL in 80 mL fresh suspension culture medium in a 250 mL Erlenmeyer flask.
    • iv.
      Culture on a shaker at 100 rpm for 2–3 days.
  • b.
    Change the fresh suspension culture medium every 2–3 days, allowing the cell density to reach 1–2×10⁷ cells/mL.
• 3.
  Freeze the suspension-cultured cells.

  • a.
    Centrifuge at 170 g for 3 min and remove the supernatant completely.

    CRITICAL: Cell viability should be ≥ 95%, and the cell density should reach about 6x10⁶ cells/mL, the cells should be in the growth phase.

  • b.
    Add the serum-free cell cryopreservation solution to adjust the cell density to about 1×10⁷ cells/mL.
  • c.
    Store at −80°C overnight.
  • d.
    Transfer to liquid nitrogen for long-term storage.

    CRITICAL: All incubation steps should be performed in a humidified incubator with 5% CO₂ at 37°C. Cell density and viability are monitored daily using a cell counter. Cell viability in the initiation process (steps 1c–1g) should be ≥ 90%, and in the maintenance process (step 2 and step 3) should be ≥ 95%.

    Pause point: Validated stable cell lines can be frozen in liquid nitrogen for long-term storage (≥2 years).

RSPO1-engineered cells in large-scale suspension culture and supernatant collection

Timing: 3–5 days

This step establishes a scalable workflow for culturing RSPO1-engineered HEK293 cells in a chemically defined, animal-source-free medium under suspension conditions, ensuring consistent cell status and sufficient cell numbers for evRSPO1 isolation.

• 4.Seed the RSPO1-engineered HEK293 cells at 1–2×10⁶ cells/mL in 80 mL of fresh suspension culture medium in a 250 mL Erlenmeyer flask.
• 5.Culture on a shaker at 100 rpm in a humidified incubator with 5% CO₂ at 37°C for 3–5 days.
• 6.When the cell density reaches 1×10⁷ cells/mL (Figure 3), the supernatant is harvested by centrifugation at 170 g for 3 min.
• 7.The cells can be passaged into fresh suspension culture medium.

Note: Cell density and viability are monitored daily using a cell counter.

CRITICAL: Low cell density and suboptimal cell condition may reduce evRSPO1 yield and quality. Optimize cell density and ensure high cell viability before collection (achieving 1×10⁷ cells/mL with >95% cell viability after 72 h of culture).

• 8.
  Supernatant pretreatment.

  • a.
    Transfer the supernatant to a new centrifuge tube and centrifuge at 300 g for 5 min to remove the dead cells.
  • b.
    Transfer the supernatant to a new centrifuge tube and centrifuge at 2,000 g for 10 min to remove the cell debris.
  • c.
    Transfer the supernatant to a new centrifuge tube and centrifuge at 10,000 g for 30 min to remove the larger vesicles.
  • d.
    Filter the supernatant through a 0.22 μm sterile filter to further remove large extracellular vesicles and residual debris.

CRITICAL: All centrifuges should be at 4°C. Incomplete removal of debris and large vesicles may reduce the purity of evRSPO1.

Note: To enhance the yield and purity of sEV, perform sequential centrifugation and filtration steps carefully before isolation.

Figure 3.

Cell viability measurement of the RSPO1-engineered HEK293 cell line in suspension culture before harvesting

(A) Representative cell density and viability when the supernatant was harvested.

(B) Quantification of cell density when the supernatant was harvested (n = 30).

(C) Quantification of cell viability when the supernatant was harvested (n = 30).

evRSPO1 isolation by EXODUS

Timing: 2–4 h per 250 mL

In this step, we describe how to produce rapid evRSPO1 isolation with the EXODUS H-600, enabling large-scale, stable, and high-purity production of functional vesicles. Approximately 7.6×10⁹ particles could be obtained per mL of supernatant, with a particle-to-protein ratio exceeding 10¹² particles/mg, indicating that our workflow could achieve high evRSPO1 yield and purity.

• 9.
  Sample Loading.

  • a.
    Add 250 mL of supernatant to the sample bottle and place it in the designated position in the adapter.
  • b.
    Mount an L-type Exodus isolation device (EID) and adapter assembly into the EID holder inside the instrument.

Note: Select the appropriate EID type based on the medium volume and membrane type: S-type EID for < 20 mL medium, M-type EID for 20–50 mL medium, and L-type EID for 50–250 mL medium.

Optional: If the culture medium volume exceeds 3 L, the EXODUS T2800 can be selected for a one-time purification.

• 10.
  Program Initialization and Run

  • a.
    Input the following parameters on-screen: sample name, sample volume, sample type (e.g., cell culture medium), and program.
  • b.
    Tap “OK” to begin the automated exosome isolation run.
• 11.
  evRSPO1 recovery

  • a.
    After completion, tap “EID OUT” on the screen to initiate EID ejection, then carefully remove it from the compartment.
  • b.
    Add 1–2 mL 1×PBS to the membrane chamber, and rinse both sides of the membrane at least 20 times each using a pipette.
  • c.
    Collect the evRSPO1-containing PBS eluate into a centrifuge tube for downstream use.

Pause point: Purified evRSPO1 can be aliquoted and stored at −80°C for up to 24 months, or at 4°C for up to 8 weeks. Avoid repeated freeze-thaw cycles.

evRSPO1 characterization and bioactivity validation

Timing: 3–5 days

This step validates the identity, concentration, purity, protein loading, and WNT signaling-inducing activity, ensuring that the purified evRSPO1s meet quality-control standards for downstream experiments.

• 12.
  Characterize the evRSPO1 particle size distribution and concentration using the NanoFCM Flow Nano Analyzer (Figure 4A).

  • a.
    Dilute evRSPO1 in sterile PBS to the optimal detection range of the instrument.
  • b.
    Acquire data for 1 min per sample, with at least 10,000 events recorded.
  • c.
    Calculate particle concentration and size distribution using the instrument’s analysis software.

Note: The recommended final concentration of diluted evRSPO1 is 10⁸–10⁹ particles/mL before detection.

CRITICAL: Preferably use the evRSPO1 produced in a single batch to minimize batch-to-batch variation for experiments in a study.

• 13.
  Assess morphology and size of evRSPO1 via negative staining with transmission electron microscopy (TEM).

  • a.
    Add 10 μL evRSPO1 suspension onto a carbon grid for 20 min.
  • b.
    Blot excess liquid with filter paper.
  • c.
    Stain the grid with 2% uranyl acetate for 1 min.
  • d.
    Air-dry the grid, then observe under TEM.

Figure 4.

Characterization of evRSPO1

(A) Size distribution of evRSPO1.

(B) TEM images of evRSPO1.

(C) Western blot analysis of RSPO1, His tag, HSP70, Calnexin, and CD63 in total cell lysate, culture supernatant (Media), large EVs, and sEVs from the RSPO1-overexpressing HEK293 cell line.

(D) Immunogold labeling analysis of evRSPO1, with red arrows indicating the RSPO1 protein.

(E) Single-molecule imaging of RSPO1 and CD63 in evRSPO1 using the dSTORM system.

Expected result: evRSPO1 exhibits a typical cup-shaped bilayer membrane structure with a diameter < 200 nm (Figure 4B).

• 14.
  Perform Western blot analysis to confirm sEV marker expression and RSPO1 loading (Figure 4C).

  • a.
    Extract total protein from sEV using a RIPA lysis buffer supplemented with protease inhibitors on ice.
  • b.
    Quantify protein concentration using the BCA assay.
  • c.
    Denature protein with 5×loading buffer at 100°C for 10 min.
  • d.
    Load about 30 μg of each protein sample into a 4%–20% SDS-PAGE gel.
  • e.
    Transfer proteins onto a PVDF membrane.
  • f.
    Block with 5% non-fat milk for 1 h at RT.
  • g.
    Incubate membranes with primary antibody (RSPO1, CD9, Calnexin, ALIX, CD63 and HSP70) at 4°C overnight.
  • h.
    Wash the membrane 3 times with TBST for 10 min each.
  • i.
    Incubate membranes with HRP-conjugated secondary antibody for 1 h at RT.
  • j.
    Wash the membrane thrice with TBST for 10 min each.
  • k.
    Detect the signal using a high-sensitivity ECL Substrate and a chemiluminescence imaging system.

CRITICAL: Load equal particle numbers of evRSPO1 and evWT for accurate comparison of RSPO1 loading.

Note: CD63 or HSP70 was used as a loading control for the sEV lysates.

• 15.
  Verify RSPO1 surface localization on evRSPO1 via immunogold labeling and TEM (Figure 4D).

  • a.
    Resuspend evRSPO1 with an equal volume of 4% paraformaldehyde for 20 min at RT.
  • b.
    Add 10 μL of the fixed evRSPO1 suspension onto a 400-mesh carbon/formvar-coated grid.
  • c.
    Allow the sample to absorb onto the grid for 20 min at RT.
  • d.
    Block the grid with 5% BSA in PBS for 10 min at RT.
  • e.
    Incubate the grid with anti-RSPO1 primary antibody for 3 h at RT.
  • f.
    Wash the grid 3 times with PBS for 5 min each.
  • g.
    Incubate the grid with 10 nm gold-conjugated secondary antibody for 1 h at RT.
  • h.
    Wash the grid 3 times with PBS for 5 min each.
  • i.
    Fix the grid with 1% glutaraldehyde in PBS for 5 min at RT.
  • j.
    Rinse the grid with ultrapure water 3 times.
  • k.
    Stain the grid with 2% uranyl acetate for 1 min at RT.
  • l.
    Air-dry the grid, then observe under TEM.
• 16.
  Single-molecule imaging of RSPO1 in evRSPO1 via dSTORM (Figure 4E).

  • a.
    Capture evRSPO1 onto coverslips pre-coated with anti-CD9, CD63, and CD81 antibodies to ensure isolation of single vesicles.
  • b.
    Label evRSPO1 with Alexa Fluor 647-conjugated anti-CD63 antibodies.
  • c.
    Incubate with anti-RSPO1 primary antibodies.
  • d.
    Alexa Fluor 488-conjugated secondary antibodies.
  • e.
    Perform sequential excitation using 640 nm and 488 nm lasers on an Abbelight SAFe 180 STORM system.

    • i.
      Acquire 10,000 frames per ROI with a 20 ms exposure time.
    • ii.
      Utilize an auto-focus lock system to minimize z-drift throughout the imaging process.
    • iii.
      Process raw data using Abbelight NEO software for super-resolution reconstruction and spatial drift correction.
• 17.
  Quantify RSPO1 loading efficiency on evRSPO1 via ELISA.

  • a.
    Pre-coat a 96-well microplate with capture antibody.
  • b.
    Incubate overnight at 4°C.
  • c.
    Aspirate the coating antibody.
  • d.
    Rinse the plate with washing buffer.
  • e.
    After blocking with 1% BSA.
  • f.
    Incubate sEV and hrRSPO1 protein samples or standards for 1.5 h at RT.
  • g.
    Remove the samples.
  • h.
    Add the protein-specific antibody and incubate for 1.5 h at RT.
  • i.
    Remove the antibody.
  • j.
    Rinse samples with the washing buffer three times.
  • k.
    Add streptavidin-HRP and incubate for 20 min at RT.
  • l.
    Remove the streptavidin-HRP.
  • m.
    Add the TMB substrate to each well.
  • n.
    Incubate for 5 min before adding the stop solution.
  • o.
    Measure the optical density using a microplate reader set to 450 nm.
• 18.
  Measure canonical WNT/β-catenin activity of evRSPO1 via TOPFlash luciferase reporter assay (Figure 5A).

  • a.
    Seed selected TOPFlash luciferase reporter assay cells in a 96-well plate at a density of 1×10⁴ cells/well in 100 μL high-glucose DMEM medium with 10% FBS and 1% PS.
  • b.
    Incubate the cells at 37°C with 5% CO₂ for 24 h.
  • c.
    Treat the cells with 1×10⁹ particles/mL evRSPO1, evWT, or 500 ng/mL hrRSPO1 for another 24 h.
  • d.
    Prepare cell lysates and measure firefly and Renilla luciferase activity using the Dual-Luciferase Reporter Assay System.
  • e.
    Determine the absorbance at 560 nm.
  • f.
    Normalize firefly luciferase activity to Renilla luciferase activity for each well.
  • g.
    Calculate the relative fold change in TOPFlash activity compared to the untreated control group.
• 19.
  Measure canonical WNT/β-catenin activity of evRSPO1 via Western blot (Figure 5B).

  • a.
    Seed HEK293 cells in a 6-well plate at a density of 3×10⁵ cells/well in 1 mL high-glucose DMEM medium with 10% FBS and 1% PS.
  • b.
    Incubate cells at 37°C with 5% CO₂ for 24 h.
  • c.
    Treat cells with 1×10⁹ particles/mL of evRSPO1, evWT, or 500 ng/mL hrRSPO1 for an additional 24 h.
  • d.
    Extract total protein from HEK293T cells using RIPA lysis buffer supplemented with 1×protease inhibitors.
  • e.
    Quantify protein concentration using a BCA assay kit.
  • f.
    Denature samples by adding 5×loading buffer and heating at 100°C for 10 min.
  • g.
    Load approximately 30 μg of protein per sample onto a 4–20% SDS-PAGE gel.
  • h.
    Transfer separated proteins onto a PVDF membrane and block with 5% non-fat milk.
  • i.
    Incubate membranes with primary antibodies (unphosphorylated β-catenin and total β-catenin) overnight at 4°C.
  • j.
    Wash thrice with TBST.
  • k.
    Incubate with HRP-conjugated secondary antibodies for 1 h at RT.
  • l.
    Detect chemiluminescent signals using a High Sensitivity ECL Substrate Kit, using β-actin as the loading control for cell lysates.
• 20.
  Validate WNT target gene upregulation via RT-qPCR.

  • a.
    Seed HEK293 cells in a 6-well plate at a density of 3×10⁵ cells/well in 1 mL high-glucose DMEM medium with 10% FBS and 1% PS.
  • b.
    Incubate cells at 37°C with 5% CO₂ for 24 h.
  • c.
    Treat cells with 1×10⁹ particles/mL evRSPO1, evWT, or 500 ng/mL hrRSPO1 for an additional 24 h.
  • d.
    Extract total RNA from the cells using TRIzol reagent.
  • e.
    Synthesize cDNA using the HiScript II 1st Strand cDNA Synthesis Kit.
  • f.
    Perform RT-qPCR using SYBR Green qPCR Master Mix, with primers for WNT target genes (AXIN2, CCND1) and the proliferation marker (Ki67), with β-actin as the reference gene.
  • g.
    Calculate relative gene expression using the 2ˆ^−ΔΔCt method.

Figure 5.

Functional validation of evRSPO1 in vitro

(A) TOPFlash reporter assay in HEK293 cells treated with evWT and evRSPO1 (n = 3).

(B) Western blot analysis of unphosphorylated β-catenin and total β-catenin in evWT- or evRSPO1-treated HEK293 cells.

(C) Representative confocal images of Axin2-mGFP expression in evWT- or evRSPO1-treated intestinal organoids.

(D) Quantification of the Axin2-mGFP signals in intestinal organoids from (C) (n = 3).

(E) Representative bright-field images of evWT- or evRSPO1-treated intestinal organoids.

(F) Quantification of the number of crypt-villus structures in intestinal organoids from (E) (n = 3). Statistical data are presented as mean ± SD. Two-tailed unpaired Student’s t-tests were used to compare the two groups. The p-values are calculated and evaluated for statistical significance (ns p ≥ 0.05 and ∗∗∗∗p < 0.0001).

In vitro functional validation using mouse intestinal organoids

Timing: 7–10 days

The following steps outline how to observe efficient uptake of evRSPO1 by intestinal organoids and validate its ability to support intestinal organoid growth, activate WNT signaling, and promote intestinal stem cell proliferation in vitro.

• 21.
  Visualization of evRSPO1 cellular uptake by intestinal organoids (Timing: 2 days).

  • a.
    Stain evRSPO1 with 10 μM PKH26 for 20 min at RT.
  • b.
    Remove the residual dye by purification again.
  • c.
    Incubate 500 μL of intestinal organoid medium containing PKH26-labeled evRSPO1 (1×10⁹ particles/mL) in a 24-well plate with intestinal organoids for 12 h.
  • d.
    Fix the organoids with 4% PFA for 30 min at RT.
  • e.
    Permeabilize the organoids with 0.3% Triton X-100 for 10 min.
  • f.
    Stain the organoids with FITC-Phalloidin and DAPI for 30 min at RT.
  • g.
    Wash the organoids 3 times with PBS.
  • h.
    Image the organoids via confocal laser scanning microscopy.
• 22.
  Treat intestinal organoids with evRSPO1.

  • a.
    Dissociate organoids into single cells via mechanical pipetting and incubation with TrypLE Select for 5 min at 37°C.
  • b.
    Centrifuge the single-cell suspension at 300 g for 3 min.
  • c.
    Resuspend the cells in Matrigel and plate in 24-well plates.
  • d.
    Incubate at 37°C for 30 min to allow Matrigel polymerization,
  • e.
    Add 500 μL organoid medium without hrRSPO1 to each well.
  • f.
    Add treatment groups to the medium: 1×10⁹ particles/mL evRSPO1, 1×10⁹ particles/mL evWT, 500 ng/mL hrRSPO1, or medium without RSPO1 (negative control).
  • g.
    Incubate the plates at 37°C with 5% CO₂ for 48 h.
• 23.
  Evaluate WNT signaling activation in organoids via fluorescence imaging of Axin2-mGFP signals (Figures 5C and 5D).

  • a.
    Treat the intestinal organoids obtained from Axin2-mGFP reporter mice with 1 × 10⁹ particles/mL of evWT or evRSPO1 for 48 h in the intestinal organoid medium without RSPO1.
  • b.
    Carefully remove the culture medium.
  • c.
    Wash the organoids once with PBS.
  • d.
    Fix the organoids with 4% PFA for 20–30 min at RT.
  • e.
    Wash with PBS twice.
  • f.
    Capture fluorescence images of the Axin2-mGFP reporter signals using a confocal microscope with consistent laser power and gain settings across all groups.
  • g.
    Quantify the Axin2-mGFP fluorescence intensity using ImageJ software.

    • i.
      Define regions of interest (ROIs) around the organoid structures.
    • ii.
      Normalize the mean fluorescence intensity to the background or to the control group.
• 24.
  Quantify organoid growth and crypt budding (Figures 5E and 5F).

  • a.
    Treat the intestinal organoids with 1×10⁹ particles/mL of evWT or evRSPO1 for 48 h in the RSPO1-removal small intestinal culture medium.
  • b.
    Capture brightfield images of treated organoids.

    • i.
      Count the number of crypt-villus budding structures per organoid, including only organoids with a diameter > 200 μm.
    • ii.
      Analyze 3 independent fields of view per biological replicate.
• 25.Perform molecular validation of WNT pathway activation and stem cell proliferation via RT-qPCR and Western blot, following the protocols in step 17 and step 18.

Note: Analyze expression of WNT target genes (Lgr5, Axin2, Myc, Ccnd1), proliferation markers (Ki67, Pcna), and cell type-specific markers (Olfm4, Ascl2, Muc2, Lyz1).

The evRSPO1 distribution in mouse intestinal tissue

Timing: 2–3 weeks

The following steps outline the in vivo observation of the biodistribution and cellular uptake of evRSPO1 in intestinal tissue after oral administration.

• 26.
  evRSPO1 labelled with DiR.

  • a.
    Stain evRSPO1 with 10 μM DiR for 20 min at RT.
  • b.
    Remove the residual dye by purification again.
• 27.
  evRSPO1 oral administration

  • a.
    Scruff the mouse firmly with the thumb and index finger, grasping the loose skin at the nape of the neck.
  • b.
    Pull the skin taut to extend the neck and align the oral cavity, pharynx, and esophagus in a straight line.
  • c.
    Insert the rounded needle tip into the mouth at the diastema, angled slightly downward along the hard palate.
  • d.
    Advance the needle slowly and gently along the posterior pharynx.
  • e.
    Gently pull back on the syringe plunger about 5 μL.
  • f.
    Correct esophageal placement: no air bubbles return, and the mouse maintains normal, quiet respiration with no gasping or cyanosis.
  • g.
    Depress the plunger slowly and steadily over 2–3 s to deliver the full volume.
  • h.
    Keep the needle angled downward during delivery to prevent solution from flowing back into the esophagus.
  • i.
    Release the mouse back into its home cage on a warmed pad. Monitor continuously for 2 min for signs of adverse events: gasping, coughing, cyanosis, lethargy, bleeding from the mouth, or abnormal posture.

Note: 20-gauge, 1.5-inch stainless steel flexible feeding needle with a rounded ball tip. 1 mL sterile tuberculin syringe; draw 200 μL labeled evRSPO1 (2×10¹⁰ particles) for each mouse. Aspiration is performed before injection to confirm proper placement (ensuring the needle is not in the trachea).

• 28.Capture the biodistribution images using the IVIS Lumina system at 2, 4, 8, and 24 h after oral administration to visualize evRSPO1 in vivo distribution.
• 29.
  Visualization of evRSPO1 cellular uptake with oral administration (Figure 6A)

  • a.
    Stain evRSPO1 with 10 μM PKH26 for 20 min at RT.
  • b.
    Remove the residual dye.
  • c.
    Treat 200 μL labeled evRSPO1 (2×10¹⁰ particles) by oral administration, following the protocols in step 25.
  • d.
    Euthanize mice via cervical dislocation 8 h after oral administration.
  • e.
    Obtain frozen-section images of the tissue using the confocal system after staining with DAPI and FITC-Phalloidin.

Figure 6.

evRSPO1 treatment promotes intestinal tissue regeneration after injury and aging

(A) Representative confocal fluorescence images of PKH26-labeled EV and FITC-labeled phalloidin in the mouse intestine after evRSPO1 treatment.

(B) Percent survival curve of radiation-induced intestinal injury in mice after evWT or evRSPO1 treatment (n = 8).

(C) Representative H&E staining of intestinal tissue from radiation-induced injury mice after evWT or evRSPO1 treatment.

(D) Quantification of the length of crypts from (C) (n = 8).

(E) Representative β-galactosidase staining of intestinal tissue from young or old mice after evWT or evRSPO1 treatment.

(F) Quantification of the positive area of β-galactosidase from (E) (n = 8).

(G) Principal component analysis (PCA) of gene expression profiles, showing sample separation by group.

(H) Heatmap showing cell cycle-related gene expression patterns in the intestinal tissue from young mice and old mice after evWT or evRSPO1 oral administration for 2 weeks. Statistical data are presented as mean ± SD. Two-tailed unpaired Student’s t-tests were used to compare the two groups. The p-values were calculated and evaluated for statistical significance (∗∗p < 0.01 and ∗∗∗∗p < 0.0001).

In vivo oral delivery of evRSPO1 for mouse intestinal tissue regeneration and injury repair

Timing: 2–3 weeks

This step validates the therapeutic efficacy of orally delivered evRSPO1 in a mouse model of radiation-induced intestinal injury, including survival monitoring and tissue repair assessment. The radiation-induced intestinal injury model was established as described by Wei et al. and Zhou et al.^6,7

• 30.
  Establish the radiation-induced intestinal injury mouse model.

  • a.
    Use 6-8-weeks-old male C57BL/6J mice, randomly divided into 3 groups: evRSPO1 treatment, evWT control, and untreated irradiation control.
  • b.
    Anesthetize mice with 2% isoflurane via inhalation.
  • c.
    Place anesthetized mice in the custom lead shielding jig, exposing only the abdominal area (from the xyphoid process to the pelvis).
  • d.
    Perform a single 12 Gy abdominal irradiation at a dose rate of 2.0 Gy/min.
  • e.
    After irradiation, provide mice with a softened diet, sterile drinking water, and soft bedding for the duration of the experiment.

CRITICAL: Ensure complete shielding of the head, thorax, limbs, and tail to avoid systemic radiation toxicity. To minimize animal stress and ensure precise positioning during the procedure, all mice are anesthetized using isoflurane before the irradiation.

• 31.
  evRSPO1 treatment by oral administration.

  • a.
    Start oral administration 1 day before irradiation, and continue daily for 5 consecutive days.
  • b.
    For each administration, orally gavage mice with 200 μL of evRSPO1 (1×10¹¹ particles/mL, total 2×10¹⁰ particles per mouse), 200 μL of evWT (equal particle number), or 200 μL sterile PBS for the untreated control.

CRITICAL: Use freshly thawed evRSPO1 for each administration to ensure maximal bioactivity.

• 32.
  Monitor mouse survival and body weight daily for 14 days post-irradiation (Figure 6B).

  • a.
    Record the number of surviving mice in each group daily.
  • b.
    Generate survival curves using the Kaplan-Meier method.
• 33.
  Collect intestinal tissue samples at designated time points.

  • a.
    Euthanize mice via cervical dislocation at days 2, 4, and 6 post-irradiation.
  • b.
    Excise the jejunum segment of the small intestine.
  • c.
    Flush the intestinal lumen with ice-cold PBS.
  • d.
    Fix a 1 cm segment of the jejunum in 4% PFA for 24 h at 4°C for histology and immunofluorescence staining.
  • e.
    Snap-freeze the remaining intestinal tissue in liquid nitrogen for RNA and protein extraction.
• 34.
  Perform histological analysis via H&E staining (Figures 6C and 6D).

  • a.
    Dehydrate the fixed intestinal tissue through an ethanol gradient and embed samples in paraffin.
  • b.
    Section the paraffin-embedded tissue at 5 μm thickness.
  • c.
    Perform H&E staining using a standard protocol.
  • d.
    Scan the stained slides using a digital slide scanner.

    • i.
      Measure crypt and villus length, and count surviving crypts per field of view via ImageJ.
    • ii.
      Analyze 5–8 random fields of view per mouse section.
• 35.
  Perform immunofluorescence staining for intestinal stem cells (ISCs), proliferation, inflammation, and tight junction markers.

  • a.
    Cryopreserve the fixed intestinal tissue in 15% sucrose solution overnight at 4°C.
  • b.
    Incubate in 30% sucrose solution until the tissue sinks to the bottom of the tube.
  • c.
    Embed the tissue in OCT compound and freeze at −80°C.
  • d.
    Section the frozen tissue at 5 μm thickness using a cryostat.
  • e.
    Permeabilize the sections with 0.3% Triton X-100 for 10 min at RT.
  • f.
    Block the sections with 3% normal sheep serum for 30 min at RT.
  • g.
    Incubate the sections with primary antibodies against Olfm4, Ki67, CD45, IFNγ, ZO-1, and Occludin at 4°C overnight.
  • h.
    Wash the sections 3 times with PBS for 5 min each.
  • i.
    Incubate the sections with fluorophore-conjugated secondary antibodies for 2 h at RT, protected from light.
  • j.
    Wash the sections 3 times with PBS for 5 min each.
  • k.
    Counterstain the sections with DAPI for 5 min at RT.
  • l.
    Mount the sections with anti-fade mounting medium.
  • m.
    Image the sections via confocal laser scanning microscopy.
  • n.
    Quantify positive cell numbers and fluorescence intensity via ImageJ.

Oral delivery of evRSPO1 for intestinal senescence reversal in aged mice

Timing: 3–4 weeks

This step validates the ability of orally delivered evRSPO1 to reverse intestinal senescence phenotypes in naturally aged mice, including activation of WNT signaling and downregulation of senescence markers.

• 36.
  Prepare aged mice for the experiment.

  • a.
    Use 18-month-old male C57BL/6J mice (aged) and 2-month-old male C57BL/6J mice (young control).
  • b.
    Randomly divide the aged mice into 2 groups: evRSPO1 treatment and evWT control.
• 37.Treat experimental mice with 200 μL of evRSPO1 (1×10¹¹ particles/mL, total 2×10¹⁰ particles per mouse) or 200 μL of evWT (equal particle number) by oral administration twice a week for 2 consecutive weeks.
• 38.Euthanize experimental mice and collect intestinal tissue samples 24 h after the last administration.
• 39.
  Detect intestinal senescence markers via β-galactosidase staining (Figures 6E and 6F).

  • a.
    Rewarm the frozen sections to RT for 10 min, then incubate with β-Galactosidase staining solution at RT for 20 min.
  • b.
    After three washes with PBS, incubate the sections with the solution at 37°C for 2 h.
  • c.
    Rinse the sections with PBS and water twice.
  • d.
    Stain the sections with Eosin-Y for 3 min.
  • e.
    Dehydrate in absolute alcohol.
  • f.
    Scan the images using a slide viewer and quantify the β-galactosidase-positive area in ImageJ.
• 40.Perform immunofluorescence staining for senescence markers (p21), ISC markers (Olfm4), proliferation marker (Ki67), and goblet cell marker (Muc2), following the protocol in step 33.
• 41.
  Perform transcriptomic analysis of intestinal tissue via RNA-seq.

  • a.
    Extract total RNA from intestinal tissue samples using TRIzol reagent.
  • b.
    Assess RNA integrity using the Bioanalyzer 2100 system.
  • c.
    Construct mRNA sequencing libraries using a standard library preparation kit.
  • d.
    Sequence the libraries on an Illumina Novaseq platform with 150 bp paired-end reads.
  • e.
    Process raw sequencing data using fastp for quality control, and map reads to the mouse reference genome using Hisat2.
  • f.
    Perform differential gene expression analysis using DESeq2.
  • g.
    Perform gene set enrichment analysis (GSEA) and gene ontology (GO) enrichment analysis using the clusterProfiler package.
• 42.Perform RT-qPCR and Western blot validation of senescence markers, WNT target genes, and gerogenes/gerosuppressors, following the protocols in step 17 and step 18.

Expected outcomes

This protocol achieves stable, scalable production of evRSPO1 with high yield and purity, which can be delivered to the intestine via oral administration. For evRSPO1 production and characterization, purified evRSPO1 will have a uniform size distribution, with > 90% of vesicles between 50–150 nm in diameter, and a particle yield of about 7.6×10⁹ particles per mL of culture supernatant. Western blot will help confirm positive expression of sEV markers, the absence of the negative marker Calnexin, and RSPO1 protein loading abundance (about 1 μg RSPO1 per 1×10¹⁰ particles). Functionally, the evRSPO1 is expected to elicit significantly higher WNT/β-catenin signaling activity, upregulate both unphosphorylated and total β-catenin protein levels, and activate canonical WNT-targeting genes, including AXIN2 and CCND1, compared to the evWT control. Critically, evRSPO1 will retain > 80% of its initial WNT activation activity after 8 weeks of storage at 4°C, whereas hrRSPO1 will show a > 70% loss of activity over the same period. In the in vitro intestinal organoid experiments, evRSPO1 will be efficiently taken up by intestinal organoids within 12 h. evRSPO1 treatment will induce strong Axin2 signals in the crypt budding regions of organoids and significantly increase the number of crypt-villus budding structures. WNT target genes (Lgr5, Axin2) and proliferation markers (Ki67, Pcna) will increase in the evRSPO1-treated organoids, while maintaining normal expression of differentiated epithelial cell markers. In in vivo mouse experiments, orally administered evRSPO1 is expected to reach the intestine and be taken up by intestinal cells, thereby inducing WNT/β-catenin signaling in crypt-resident cells. In injury models, evRSPO1 administration is expected to improve survival and accelerate crypt recovery and epithelial regeneration. Overall, this protocol provides a reproducible platform for delivering RSPO1 via sEVs and evaluating the therapeutic efficacy of the delivery systems in both in vitro and in vivo settings.

Limitations

This protocol has several important limitations that should be considered when executing the experiments and interpreting the results. First, the efficiency of RSPO1 loading onto sEVs depends on its interaction with heparan sulfate proteoglycans (HSPGs), which may vary across cell types and culture conditions. As a result, reproducibility of protein loading and batch-to-batch consistency may require careful optimization and validation. Second, the protocol is optimized for laboratory-scale production of evRSPO1 using suspension culture in shaker flasks; scale-up to industrial bioreactor production requires additional process development and validation, including optimization of culture parameters, harvest timing, and isolation workflows to maintain consistent product quality. Third, while this protocol demonstrates robust activity in intestinal organoids and mouse models, its applicability to other tissues or disease models has not been extensively validated. Additional optimization may be required when extending this approach to different biological systems. Finally, the protocol uses HEK293 cell-derived sEV, which are well-characterized for preclinical research; however, clinical-grade production requires compliance with GMP guidelines, including full characterization of the master cell bank, viral safety testing, and comprehensive product release testing, which are not covered in this laboratory-scale protocol.

Troubleshooting

Problem 1

Low yield of evRSPO1 production.

Potential solution

Optimize cell density and ensure high cell viability before collection (cell density reached 1×10⁷ cells/mL with > 95% cell viability over 72 h of culture) (step 4).

Problem 2

Low purity of evRSPO1 production.

Potential solution

Carefully perform sequential centrifugation and filtration steps to remove cellular debris and larger vesicles before isolation (step 6).

Problem 3

Low protein loading efficiency of RSPO1 on evRSPO1.

Potential solution

Increase the number of lentiviral infections in the HEK293 cells (Preparation of RSPO1 protein-overexpressing HEK293 cells). Optimize culture conditions to maintain protein integrity and verify that RSPO1 is expressed in producer cells by Western blot (step 12).

Problem 4

Low cell viability of evRSPO1 producer cells.

Potential solution

Optimize culture conditions to maintain cell viability (steps 1–3). Adjust the volume and rotation speed of the culture medium in the shake flask. If necessary, replace the fresh culture medium every two days (step 4).

Problem 5

Low TOPFlash activity of purified evRSPO1.

Potential solution

Store evRSPO1 at −80°C for long-term (up to 2 years) and avoid repeated freeze-thaw cycles. For long-term storage, consider aliquoting samples to maintain consistency. For short-term use, evRSPO1 dilution in the medium can be stored up to 2 weeks (Part 3: Pause point).

Problem 6

Poor intestinal organoid growth after evRSPO1 treatment.

Potential solution

Confirm RSPO1 presence and activity using a TOPFlash reporter assay. Ensure that the organoids are in good condition. Optimize particle concentration and exposure time in the organoid (step 20).

Problem 7

No WNT signaling activation in the mouse intestine after oral evRSPO1 administration.

Potential solution

Ensure consistent oral gavage technique and dosing volume. Avoid using evRSPO1 that undergoes repeated freeze-thaw cycles. Use freshly prepared or properly labeled evRSPO1 for tracking. Optimize administration frequency and particle number based on experimental design (step 25).

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yun-Shen Chan (chan_yunshen@gzlab.ac.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Lingyan Yang (yang_lingyan@gzlab.ac.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

All data presented are derived from the same experiments as reported in Yang et al.^1,2 RNA-seq data can be obtained from the public database (SRA data: PRJNA1508718).

Acknowledgments

This work was funded by the National Natural Science Foundation of China (82370053), Guangzhou National Laboratory Fund (GZNL2025C02009), and the Guangdong Provincial Pearl River Talents program (2023QN10Y489).

Author contributions

L.Y. designed and optimized the HEK293 suspension culture protocols and engineered evRSPO1 purification protocols, designed the study, and drafted the article. T.J. optimized the intestinal organoid culture and in vitro evRSPO1 function verification protocols and drafted the article. S.X. optimized the HEK293 engineered protocols. Y.Y. optimized the evRSPO1 oral administration protocols. Z.Q. optimized the verification protocols for the aged mouse model. Y.-S.C. served as scientific advisor and critically reviewed the protocols.

Declaration of interests

L.Y. and Y.-S.C. have filed patents related to technologies used in the present work.