The Argentine powerhouse hold a two-goal advantage after accounting for Libertad 3-1 in the opening leg of their last-16 tie in Paraguay last week.
Despite losing leading goalscorer Teofilo Gutierrez and Eder Alvarez Balanta to Colombia national duty, and Leonel Vangioni to Argentina, River still emerged triumphant after a three-goal second half at the Estadio Dr Nicolas Leoz.
The trio will return to the River line-up for the return leg in Buenos Aires after playing pivotal roles in Sunday's comprehensive 3-0 victory over Belgrano in Argentina Primera Division.
"We have to be very smart. I need all the players; see how they recover and last minute we'll see how we go," Gallardo told River's official website following Sunday's win.
"There are players who stand out because the quality of each is different but when the whole operation achieves a good talk about how good the team.
"The mentality is strong and the desire to want to play well too. Good to end a round with a good win and performance."
But Penarol - unbeaten in 12 matches at home in all competitions - will take plenty of confidence into Wednesday's fixture, with Estudiantes only able to register one win in seven competitive games.
"We had the intention of getting a result at home and did not but we have to go find the result away from home," Arruabarrena told reporters after Sunday's 3-2 win at Godoy Cruz.
"We know it will be tough but we will go and try."
Meanwhile, Paraguay's Cerro Porteno travel to Lanus on Tuesday protecting a 2-1 lead.
